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.

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.

Development of gene expression system in egg cells and zygotes isolated from rice and maize.

Polyethylene glycol calcium (PEG-Ca2+) transfection-mediated analysis allows rapid and efficient examination of gene function. To investigate the diverse cellular functions of genes of interest in plant cells, macromolecules, such as DNA, RNA, and proteins, are delivered into protoplasts prepared from somatic tissues or calli using a PEG-Ca2+ transfection procedure. To take advantage of this macromolecule delivery system in the reproductive and developmental biology of angiosperms, this study established a PEG-Ca2+ transfection system with isolated egg cells and zygotes. The conditions for PEG and plasmid DNA concentrations for transfection of rice egg cells were first addressed, and ~30% of PEG-Ca2+-transfected egg cells showed exogenous and transient expressions of fluorescent proteins from plasmid DNA delivered into the cells. Interestingly, a dual expression of two different fluorescent proteins in the same egg cell using two kinds of plasmid DNAs was also observed. For PEG-Ca2+ transfection with maize zygotes, ~80% of zygotes showed expression of GFP proteins from plasmid DNA. Importantly, PEG-transfected zygotes developed normally into cell masses and mature plants. These results suggest that the present PEG-Ca2+-mediated transient expression system provides a novel and effective platform for expressing and analyzing genes of interest in egg cells and zygotes. Moreover, combined with the CRISPR/Cas9 approach, the present transient expression system in zygotes will become a powerful and alternative tool for the preparation of gene-edited plants.