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Live‐cell imaging of the chloroplast outer envelope membrane using fluorescent dyes

Chloroplasts are organelles composed of sub‐organellar compartments—stroma, thylakoids, and starch granules—and are surrounded by outer and inner envelope membranes (OEM and IEM, respectively). The chloroplast OEM and IEM play key roles not only as a barrier separating the chloroplast components fro...

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Autores principales: Ichikawa, Shintaro, Ishikawa, Kazuya, Miyakawa, Hitoshi, Kodama, Yutaka
Formato: Online Artículo Texto
Lenguaje:English
Publicado: John Wiley and Sons Inc. 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9666008/
https://www.ncbi.nlm.nih.gov/pubmed/36398034
http://dx.doi.org/10.1002/pld3.462
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author Ichikawa, Shintaro
Ishikawa, Kazuya
Miyakawa, Hitoshi
Kodama, Yutaka
author_facet Ichikawa, Shintaro
Ishikawa, Kazuya
Miyakawa, Hitoshi
Kodama, Yutaka
author_sort Ichikawa, Shintaro
collection PubMed
description Chloroplasts are organelles composed of sub‐organellar compartments—stroma, thylakoids, and starch granules—and are surrounded by outer and inner envelope membranes (OEM and IEM, respectively). The chloroplast OEM and IEM play key roles not only as a barrier separating the chloroplast components from the cytosol but also in the interchange of numerous metabolites and proteins between the chloroplast interior and the cytosol. Fluorescent protein markers for the chloroplast OEM have been widely used to visualize the outermost border of chloroplasts. However, the use of marker proteins requires an established cellular genetic transformation method, which limits the plant species in which marker proteins can be used. Moreover, the high accumulation of OEM marker proteins often elicits abnormal morphological phenotypes of the OEM. Because the OEM can currently only be visualized using exogenous marker proteins, the behaviors of the chloroplast and/or its OEM remain unknown in wild‐type cells of various plant species. Here, we visualized the OEM using live‐cell staining with the fluorescent dyes rhodamine B and Nile red in several plant species, including crops. We propose rhodamine B and Nile red as new tools for visualizing the chloroplast OEM in living plant cells that do not require genetic transformation. SIGNIFICANCE STATEMENT: We established a live‐cell imaging method to visualize the chloroplast outer envelope membrane by staining living cells with fluorescent dyes. This method does not require genetic transformation and allows the observation of the chloroplast outer envelope membrane in various plant species.
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spelling pubmed-96660082022-11-16 Live‐cell imaging of the chloroplast outer envelope membrane using fluorescent dyes Ichikawa, Shintaro Ishikawa, Kazuya Miyakawa, Hitoshi Kodama, Yutaka Plant Direct Original Research Chloroplasts are organelles composed of sub‐organellar compartments—stroma, thylakoids, and starch granules—and are surrounded by outer and inner envelope membranes (OEM and IEM, respectively). The chloroplast OEM and IEM play key roles not only as a barrier separating the chloroplast components from the cytosol but also in the interchange of numerous metabolites and proteins between the chloroplast interior and the cytosol. Fluorescent protein markers for the chloroplast OEM have been widely used to visualize the outermost border of chloroplasts. However, the use of marker proteins requires an established cellular genetic transformation method, which limits the plant species in which marker proteins can be used. Moreover, the high accumulation of OEM marker proteins often elicits abnormal morphological phenotypes of the OEM. Because the OEM can currently only be visualized using exogenous marker proteins, the behaviors of the chloroplast and/or its OEM remain unknown in wild‐type cells of various plant species. Here, we visualized the OEM using live‐cell staining with the fluorescent dyes rhodamine B and Nile red in several plant species, including crops. We propose rhodamine B and Nile red as new tools for visualizing the chloroplast OEM in living plant cells that do not require genetic transformation. SIGNIFICANCE STATEMENT: We established a live‐cell imaging method to visualize the chloroplast outer envelope membrane by staining living cells with fluorescent dyes. This method does not require genetic transformation and allows the observation of the chloroplast outer envelope membrane in various plant species. John Wiley and Sons Inc. 2022-11-15 /pmc/articles/PMC9666008/ /pubmed/36398034 http://dx.doi.org/10.1002/pld3.462 Text en © 2022 The Authors. Plant Direct published by American Society of Plant Biologists and the Society for Experimental Biology and John Wiley & Sons Ltd. https://creativecommons.org/licenses/by/4.0/This is an open access article under the terms of the http://creativecommons.org/licenses/by/4.0/ (https://creativecommons.org/licenses/by/4.0/) License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.
spellingShingle Original Research
Ichikawa, Shintaro
Ishikawa, Kazuya
Miyakawa, Hitoshi
Kodama, Yutaka
Live‐cell imaging of the chloroplast outer envelope membrane using fluorescent dyes
title Live‐cell imaging of the chloroplast outer envelope membrane using fluorescent dyes
title_full Live‐cell imaging of the chloroplast outer envelope membrane using fluorescent dyes
title_fullStr Live‐cell imaging of the chloroplast outer envelope membrane using fluorescent dyes
title_full_unstemmed Live‐cell imaging of the chloroplast outer envelope membrane using fluorescent dyes
title_short Live‐cell imaging of the chloroplast outer envelope membrane using fluorescent dyes
title_sort live‐cell imaging of the chloroplast outer envelope membrane using fluorescent dyes
topic Original Research
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9666008/
https://www.ncbi.nlm.nih.gov/pubmed/36398034
http://dx.doi.org/10.1002/pld3.462
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