Transcriptome analyses uncover reliance of endosperm gene expression on Arabidopsis embryonic development.

Endosperm-embryo development in flowering plants is regulated coordinately by signal exchange during seed development. However, such a reciprocal control mechanism has not been clearly identified. In this study, we identified an endosperm-specific gene, LBD35, expressed in an embryonic development-dependent manner, by a comparative transcriptome and cytological analyses of double-fertilized and single-fertilized seeds prepared by using the kokopelli mutant, which frequently induces single fertilization events. Transcriptome analysis using LBD35 as a marker of the central cell fertilization event identified that 141 genes, including 31 genes for small cysteine-rich peptides, are expressed in a double fertilization-dependent manner. Our results reveal possible embryonic signals that regulate endosperm gene expression and provide a practicable method to identify genes involved in the communication during endosperm-embryo development.

Regulatory functions of ROS dynamics via glutathione metabolism and glutathione peroxidase activity in developing rice zygote.

Reactive oxygen species (ROS) play essential roles in plant development and environmental stress responses. In this study, ROS dynamics, the glutathione redox status, the expression and subcellular localization of glutathione peroxidases (GPXs), and the effects of inhibitors of ROS-mediated metabolism were investigated along with fertilization and early zygotic embryogenesis in rice (Oryza sativa). Zygotes and early embryos exhibited developmental arrest upon inhibition of ROS production. Egg cells accumulated high ROS levels, and, after fertilization, intracellular ROS levels progressively declined in zygotes in which de novo expression of GPX1 and 3 was observed through upregulation of the genes. In addition to inhibition of GPX activity, depletion of glutathione impeded early embryonic development and led to failure of the zygote to appropriately decrease H2 O2 levels. Moreover, through monitoring of the glutathione redox status, the developing zygotes exhibited a progressive glutathione oxidation, which became extremely delayed under inhibited GPX activity. Our results provide insights into the importance of ROS dynamics, GPX antioxidant activity, and glutathione redox metabolism during zygotic/embryonic development.

Effect of Paternal Genome Excess on the Developmental and Gene Expression Profiles of Polyspermic Zygotes in Rice.

Polyploid zygotes with a paternal gamete/genome excess exhibit arrested development, whereas polyploid zygotes with a maternal excess develop normally. These observations indicate that paternal and maternal genomes synergistically influence zygote development via distinct functions. In this study, to clarify how paternal genome excess affects zygotic development, the developmental and gene expression profiles of polyspermic rice zygotes were analyzed. The results indicated that polyspermic zygotes were mostly arrested at the one-cell stage after karyogamy had completed. Through comparison of transcriptomes between polyspermic zygotes and diploid zygotes, 36 and 43 genes with up-regulated and down-regulated expression levels, respectively, were identified in the polyspermic zygotes relative to the corresponding expression in the diploid zygotes. Notably, OsASGR-BBML1, which encodes an AP2 transcription factor possibly involved in initiating rice zygote development, was expressed at a much lower level in the polyspermic zygotes than in the diploid zygotes.

In Vitro Production of Zygotes by Electrofusion of Rice Gametes.

In angiosperms, fertilization and embryogenesis occur in the embryo sac, which is deeply embedded in ovular tissue. In vitro fertilization (IVF) systems using isolated gametes have been utilized to dissect postfertilization events in angiosperms, such as egg activation, zygotic development, and early embryogenesis. In addition, using IVF systems, interspecific zygotes and polyploid zygotes have been artificially produced, and their developmental profiles/mechanisms have been analyzed. Taken together, the IVF system can be considered a powerful technique for investigating the fertilization-induced developmental sequences in zygotes and generating new cultivars with desirable characteristics. Here, we describe the procedures for the isolation of rice gametes, electrofusion of gametes, and the culture of the produced zygotes and embryo.

Polyspermy in angiosperms: Its contribution to polyploid formation and speciation.

Polyploidization has played a major role in the long-term diversification and evolutionary success of angiosperms. Triploid formation among diploid plants, which is generally considered to be achieved by fertilization of an unreduced gamete with a reduced one, has been accepted as a means of polyploid production. In addition, it has been supposed that polyspermy also contributes to the triploid formation in maize, wheat, and some orchids; however, such a mechanism has been considered uncommon because reproducing the polyspermic situation and unambiguously investigating developmental profiles of polyspermic zygotes are difficult. To overcome these problems, rice polyspermic zygotes have been successfully produced by electrofusion of an egg cell with two sperm cells, and their developmental profiles have been monitored. The triploid zygotes progress through karyogamy and divide into two-celled embryos via a typical bipolar mitotic division; the two-celled embryos further develop into triploid plants, indicating that polyspermic plant zygotes, unlike those of animals, can develop normally. Furthermore, progenies consisting of triparental genetic materials have been successfully obtained in Arabidopsis through the pollination of two different kinds of male parents with a female parent. These different pieces of evidence for development and emergence of polyspermic zygotes in vitro and in planta suggest that polyspermy is a key event in polyploidization and species diversification.

An efficient DNA- and selectable-marker-free genome-editing system using zygotes in rice.

Technology involving the targeted mutagenesis of plants using programmable nucleases has been developing rapidly and has enormous potential in next-generation plant breeding. Notably, the clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein-9 nuclease (Cas9) (CRISPR-Cas9) system has paved the way for the development of rapid and cost-effective procedures to create new mutant populations in plants1,2. Although genome-edited plants from multiple species have been produced successfully using a method in which a Cas9-guide RNA (gRNA) expression cassette and selectable marker are integrated into the genomic DNA by Agrobacterium tumefaciens-mediated transformation or particle bombardment3, CRISPR-Cas9 integration increases the chance of off-target modifications4, and foreign DNA sequences cause legislative concerns about genetically modified organisms5. Therefore, DNA-free genome editing has been developed, involving the delivery of preassembled Cas9-gRNA ribonucleoproteins (RNPs) into protoplasts derived from somatic tissues by polyethylene glycol-calcium (PEG-Ca2+)-mediated transfection in tobacco, Arabidopsis, lettuce, rice6, Petunia7, grapevine, apple8 and potato9, or into embryo cells by biolistic bombardment in maize10 and wheat11. However, the isolation and culture of protoplasts is not feasible in most plant species and the frequency of obtaining genome-edited plants through biolistic bombardment is relatively low. Here, we report a genome-editing system via direct delivery of Cas9-gRNA RNPs into plant zygotes. Cas9-gRNA RNPs were transfected into rice zygotes produced by in vitro fertilization of isolated gametes12 and the zygotes were cultured into mature plants in the absence of selection agents, resulting in the regeneration of rice plants with targeted mutations in around 14-64% of plants. This efficient plant-genome-editing system has enormous potential for the improvement of rice as well as other important crop species.

An imbalanced parental genome ratio affects the development of rice zygotes.

Upon double fertilization, one sperm cell fuses with the egg cell to form a zygote with a 1:1 maternal-to-paternal genome ratio (1m:1p), and another sperm cell fuses with the central cell to form a triploid primary endosperm cell with a 2m:1p ratio, resulting in formation of the embryo and the endosperm, respectively. The endosperm is known to be considerably sensitive to the ratio of the parental genomes. However, the effect of an imbalance of the parental genomes on zygotic development and embryogenesis has not been well studied, because it is difficult to reproduce the parental genome-imbalanced situation in zygotes and to monitor the developmental profile of zygotes without external effects from the endosperm. In this study, we produced polyploid zygotes with an imbalanced parental genome ratio by electro-fusion of isolated rice gametes and observed their developmental profiles. Polyploid zygotes with an excess maternal gamete/genome developed normally, whereas approximately half to three-quarters of polyploid zygotes with a paternal excess showed developmental arrests. These results indicate that paternal and maternal genomes synergistically serve zygote development with distinct functions, and that genes with monoallelic expression play important roles during zygotic development and embryogenesis.

Development of polyspermic zygote and possible contribution of polyspermy to polyploid formation in angiosperms.

Fertilization is a general feature of eukaryotic uni- and multicellular organisms to restore a diploid genome from female and male gamete haploid genomes. In angiosperms, polyploidization is a common phenomenon, and polyploidy would have played a major role in the long-term diversification and evolutionary success of plants. As for the mechanism of formation of autotetraploid plants, the triploid-bridge pathway, crossing between triploid and diploid plants, is considered as a major pathway. For the emergence of triploid plants, fusion of an unreduced gamete with a reduced gamete is generally accepted. In addition, the possibility of polyspermy has been proposed for maize, wheat and some orchids, although it has been regarded as an uncommon mechanism of triploid formation. One of the reasons why polyspermy is regarded as uncommon is because it is difficult to reproduce the polyspermy situation in zygotes and to analyze the developmental profiles of polyspermic triploid zygotes. Recently, polyspermic rice zygotes were successfully produced by electric fusion of an egg cell with two sperm cells, and their developmental profiles were monitored. Two sperm nuclei and an egg nucleus fused into a zygotic nucleus in the polyspermic zygote, and the triploid zygote divided into a two-celled embryo via mitotic division with a typical bipolar microtubule spindle. The two-celled proembryos further developed and regenerated into triploid plants. These suggest that polyspermic plant zygotes have the potential to form triploid embryos, and that polyspermy in angiosperms might be a pathway for the formation of triploid plants.

Formation of triploid plants via possible polyspermy.

Polyploidization is a common phenomenon in angiosperms, and polyploidy has played a major role in the long-term diversification and evolutionary success of plants. Triploid plants are considered as the intermediate stage in the formation of stable autotetraploid plants, and this pathway of tetraploid formation is known as the triploid bridge. As for the mechanism of triploid formation among diploid populations, fusion of an unreduced gamete with a reduced gamete is generally accepted. In addition, the possibility of polyspermy has been proposed for maize, wheat and some orchids, although it has been regarded as an uncommon mechanism of polyploid formation. One of the reasons why polyspermy is regarded as uncommon is because it is difficult to reproduce the polyspermy situation in zygotes and to analyze the developmental profiles of polyspermic zygotes. In the study, we produced polyspermic rice zygotes by electric fusion of an egg cell with two sperm cells and monitored their developmental profiles. The two sperm nuclei and the egg nucleus fused into a zygotic nucleus in the polyspermic zygote, and the triploid zygote divided into a two-celled embryo via mitotic division with a typical bipolar microtubule spindle. The two-celled proembryos developed and regenerated into triploid plants. These results suggest that polyspermic plant zygotes have the potential to form triploid embryos, and that polyspermy in angiosperms might be a pathway for the formation of triploid plants.