Plants

Elucidating metabolites during plant cell division | RIKEN

March 18, 2022

Riken

-1 High-precision capture of cellular content through cellular analysis-

Emiko Okubo (Kurihara), Researcher, Director, Minami Matsui Group, Synthetic Genomics Research Group, Environmental Resources Science Research Center, Institute of Physics and Chemistry (RIKEN), Ahmed Ali Intern (Research), Single Cell Mass Analysis Research Group (while researching ), Center for Living Systems Research, then research teams such as the current Center for Biofunctional Science, Visiting Investigator, Cell-Free Protein Synthesis Research Team) and Takayuki Kawai (at the time of research, current Research Center for Biofunctional Science, Visiting Investigator, no Cellular Protein Synthesis Research Group) has plant cells.[1]We elucidate fluctuations in intracellular metabolites in subphases (prophase, metaphase, anaphase, and telophase) at the single-cell level.

The result of this research is the “cell plate.”[2]Not only helps to elucidate the mechanism of mitosis in plant cells with insertions, but also contributes to plant biomass[3]It can be expected to help create useful technology.

Information on the metabolites that make up mitotic cells is expected to reveal important factors in mitotic progression and the mechanism of cell plate insertion, but it corresponds to each subphase of mitosis, and the composition of intracellular metabolites is unclear.

This time, the team analyzed the mitotic metabolome of a plant using cultured tobacco cells with visualized chromosomes.[4]We succeeded in elucidating the fluctuation of intracellular metabolites during mitosis.

This study is based on scientific journals”plant physiologyPublished in the online edition (March 18: March 18 Japan time).

Plant cell mitotic phase diagram and intracellular metabolite subphase segregation diagram

Image of plant cells during mitosis and subphase separation of intracellular metabolites

background

Eukaryotic mitosis is the cell cycle[5]It executes in M ​​cycles of G1, S, G2 and M cycles.Two sister chromatids during mitosis[6]is an important process for accurate and uniform distribution to both daughter cells. Mitosis is divided into prophase, metaphase, anaphase and telophase subphase, the chromosomes in the prophase are condensed, and the chromosomes in the metaphase are arranged on the equatorial plane. Chromosomes then segregate at anaphase and sister chromatids decondensate at the final stage.

During mitosis in plant cells, a “cell plate” (new cell wall) begins to insert between daughter nuclei that divide into poles at the end of the cycle, and subsequent cytokinesis completes cell division. In plants with strong cell walls surrounding the cells, the normal insertion of the cell plate into the division plane allows for an accurate and even distribution of chromosomes and cytoplasmic components.Chromosome and cytoskeleton based on previous imaging analysis[7]small cells[7]It has been elucidated that intracellular structures such as these complete the insertion of the cell plate (cell division) while adopting coordinated and dynamic dynamics throughout the division phase.

Therefore, information on the substances that make up cells should also be tightly controlled, and it is expected that, if known, important factors in the progression of mitosis and the mechanism of insertion into the cell plate will be elucidated. However, the composition of intracellular metabolites corresponding to prophase, metaphase, anaphase, and telophase remains unclear. This is because the mitotic stages cannot be completely synchronized, and methods for collecting large numbers of cells involve information about cells in other substages. Therefore, the research team’s goal was to establish a method to obtain and analyze cells in prophase, metaphase, anaphase and telophase.

Research Methods and Results

First, the research team visualized the nuclei (chromosomes) of tobacco BY-HR cells to observe prophase, metaphase, anaphase, and telophase in detail.[8]established (A in the figure below).Wall-free protoplasts from BY-HR cells to obtain only one cell of interest[9]Cells corresponding to each of prophase, metaphase, anaphase, and telophase were prepared and manually obtained using a robotic arm in glass capillaries, depending on the state of the chromosome.Glass Capillary Mass Spectrometer[10]Estimate and quantify intracellular metabolites with direct setup.

It was found that metabolites in prophase, midphase, late phase, and telophase exhibited time-specific accumulation patterns and were tightly controlled (Figure B below). In addition, it was revealed that a large amount of macromolecular metabolites were detected before the observation of the cell plate, i.e., late stage, and decreased in the final stage (lower panel C). Furthermore, we were able to elucidate the increased high molecular weight lipids in late and late stages.

Subphase separation and characterization of intracellular metabolites

Separation and characterization of intracellular metabolite subphases

  • A:Time-lapse images of chromosomes and the cytoskeleton during mitosis were obtained with confocal laser scanning microscopy. Plates of cells form and develop centrifugally in late to late stages within structures called fragmented microtubules.
  • Second:The accumulation pattern of metabolites is different in each subphase.
  • C:The amount of macromolecular metabolites contained in each period increased in the later period and decreased in the end period.

future expectations

In this study, we successfully captured the intracellular metabolites of each mitotic subphase and took a step towards understanding their role in plant cell mitosis. In the future, we plan to carry out this study to identify metabolites that are critical for the progression of mitosis and normal cell division.

The cell plate inserted during plant cell division is the newly formed cell wall.Cellulose is the main component of plant cell walls, while bioethanol[3]It is a resource that becomes a raw material, eg. The results of this study contributed to plant biomass research in terms of cell number control and material production, and as a result, the United Nations declared the Sustainable Development Goals (SDGs) in 2016 as clean energy research.[11]Among them, it can be expected to contribute to “7. Energy and cleanliness for all”.

Furthermore, if we perform single-cell metabolome analysis not only in plant cells, but also in various species, it is believed that part of the common control mechanism of mitosis will be elucidated.

Supplementary Instructions

  • 1.Ito split
    A mode of eukaryotic cell division that refers to the M phase of the G1, S, G2, and M phases of the cell cycle. The process of mitosis (M phase) is divided into four sub-phases: prophase, metaphase, anaphase and telophase according to the state of chromosomes. Chromosome condensation occurs in the early stage, the chromosomes are arranged on the equatorial plane in the middle stage, the chromosomes separate in the late stage, and finally the chromosome decondensation occurs.
  • 2.cell plate
    During cytokinesis, which occurs after plant cell mitosis, new cell wall structures are formed between the separated daughter cells. During the later stages of cell division, fragmented plastids (phragmoplasts) are formed that transport supplies to the cell plate and support cell plate development. The cell plate emerges from its central part (telophase), develops in coordination with the membranous body, disappears during cytokinesis, fuses with the primitive cell wall, and divides the cell into two minutes.
  • 3.Plant biomass, bioethanol
    Plant biomass is a concept representing the amount of plant resources, and is a renewable plant-derived resource that does not include fossil resources. Bioethanol (biomass ethanol) is mainly produced by fermenting glucose contained in plant resources. Cellulose is a component of plant cell walls, and is also saccharified and fermented to become the raw material for bioethanol.
  • 4.Metabolome Analysis
    Metabolome is the general term for metabolites contained in the body. Metabolome analysis is to comprehensively elucidate the types and concentrations of metabolites contained. Data is acquired by measuring the sample with a mass spectrometer. It also plays an important role in linking genomic information, gene expression information and phenotypic information.
  • 5.cell cycle
    In eukaryotes, a series of phenomena in which a cell divides into two daughter cells and increases after division. From microscopic observation, it is divided into interphase (G1, S, G2) and M phase, that is, mitosis. The cell cycle is one week and proceeds around the G1, S, G2 and M phases. When proliferation ceases, the stationary phase (G0) is entered.
  • 6.sister chromatids
    Each pair of duplicated chromosomes is called a sister chromatid. The metaphase chromosomes are aligned on the equatorial plane, and the anaphase chromosomes are divided into two, moving to the poles, each called a sister chromatid.
  • 7.Cytoskeleton, vesicles
    The cytoskeleton mainly refers to actin fibers, intermediate filaments and microtubules, and is an intracellular structure involved in the deformation and migration of membrane organelles, such as cell morphology and intracellular vacuolar membranes and vacuoles. .. it is also closely related to cell division and cell plate insertion. Vesicles are structures surrounded by cell membranes, and plants contain various organelles such as vacuoles and endoplasmic reticulum.
  • 8.Tobacco BY-HR cells
    In the study of plant cell cycle, cultured tobacco cells are often used because of their high growth efficiency, easy cell observation, and ability to perform synchronous culture. In this study, BY-HR cells (tobacco BY-2 cells expressing histone H2B-RFP) were created as cultured tobacco cells whose chromosomes were labeled with fluorescent proteins.
  • 9.protoplast
    Plant cells in which the cell walls of the plant cells are cleaved by enzymatic treatment. In this study, it was used to isolate cultured tobacco cells in units of several cells.
  • 10.mass analyzer
    A device that ionizes the atoms and molecules that make up a substance, separates the ions generated in the vacuum container according to the mass-to-charge ratio (m/z), and detects and measures the mass number and quantity. Also called a mass spectrometer.
  • 11.Sustainable Development Goals (SDGs)
    International goals for the period 2016 to 2030 are set out in the 2030 Agenda for Sustainable Development, adopted at the United Nations summit in September 2015. Composed of 17 goals and 169 goals, the realization of a sustainable world is a common thing, not only developing countries, but also developed countries themselves are working hard, and Japan is also actively working on it. (Reprinted from the website of the Ministry of Foreign Affairs with some modifications).

research team

Riken
RIKEN Center for Questionable Genomics Research
Researcher Okubo-Kurihara Emiko
Intern (while researching) Mika Hiramoto
Senior Researcher Shio Kurihara (Yukio Kurihara)
Group Director Minami Matsui
Living Systems Research Center (at the time of research) Single-cell mass spectrometry research team (at the time of research)
Intern (while researching) Ahmed Ali
(Currently Assistant Professor of Leiden University, Visiting Researcher of Cell-Free Protein Synthesis Research Group of RIKEN Biosystems Innovation Center)
Researcher (at the time of research) Kawai Takayuki
(Currently Associate Professor, Faculty of Science, Kyushu University, Visiting Researcher, Cell Protein Synthesis Research Group, Center for Biosystems Bioscience, Institute of Physics and Chemistry)
Team leader (during research) Masujima Tsutomu

research support

The research was conducted by the RIKEN Foundation “1 Cell Project (Research Partners: Emiko Kurihara)”, RIKEN Pioneering Projects “Biology of the Intracellular Environment (Research Partners: Shio Kurihara, Minami Matsui)” and the Japan Society for the Promotion of Science (JSPS). Scientific Research Grant Grant Challenging Research (Sprouting) This was conducted with the support of “Single-cell omics analysis of plant cell division stages (Research Lead: Emiko Kurihara)”.

Base paper information

  • Okubo-Kurihara, E *., Ali, A *., Hiramoto, M., Kurihara, Y., Abouleila, Y., Abdelazem, E., Kawai, T., Makita, Y., Kawashima, M., Esaki , T., Shimada, H., Mori, T., Hirai, M., Higaki, T., Hasezawa, S., Shimizu, Y., Masujima, T., Matsui, M. “Tracking single-cell metabolite- Resolution reveals metabolic dynamics during plant mitosis”, plant physiology *These authors contributed equally.

host

Riken
RIKEN Center for Questionable Genomics Research
Researcher Okubo-Kurihara Emiko
Group Director Minami Matsui
Living Systems Research Center (at the time of research) Single-cell mass spectrometry research team (at the time of research)
Intern (while researching) Ahmed Ali
(currently a visiting researcher in the Cell-Free Protein Synthesis Research Group, Biosystems Innovation Center, Institute of Physics and Chemistry)
Researcher (at the time of research) Kawai Takayuki
(currently a visiting researcher in the Cell-Free Protein Synthesis Research Group, Biosystems Innovation Center, Institute of Physics and Chemistry)

Photo of researcher Emiko Okubo (Kurihara)
Emiko Okubo (Kurihara)
Photo of Ahmed Ali intern (during research)
Ahmed Ali

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