Imaging of the Entry Pathway of a Cell-Penetrating Peptide–DNA Complex From the Extracellular Space to Chloroplast Nucleoids Across Multiple Membranes in Arabidopsis Leaves

Each plant cell has hundreds of copies of the chloroplast genome and chloroplast transgenes do not undergo silencing. Therefore, chloroplast transformation has many powerful potential agricultural and industrial applications. We previously succeeded in integrating exogenous genes into the chloroplas...

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Autores principales:	Kazusato Oikawa, Ayaka Tateishi, Masaki Odahara, Yutaka Kodama, Keiji Numata
Formato:	article
Lenguaje:	EN
Publicado:	Frontiers Media S.A. 2021
Materias:	Arabidopsis thaliana cell-penetrating peptide DNA delivery chloroplast nucleoid field emission-scanning electron microscope confocal laser scanning microscopy Plant culture SB1-1110
Acceso en línea:	https://doaj.org/article/c91ced86e1cc4e60ad3cff00e7db8b45
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spelling	oai:doaj.org-article:c91ced86e1cc4e60ad3cff00e7db8b452021-12-03T06:23:18ZImaging of the Entry Pathway of a Cell-Penetrating Peptide–DNA Complex From the Extracellular Space to Chloroplast Nucleoids Across Multiple Membranes in Arabidopsis Leaves1664-462X10.3389/fpls.2021.759871https://doaj.org/article/c91ced86e1cc4e60ad3cff00e7db8b452021-12-01T00:00:00Zhttps://www.frontiersin.org/articles/10.3389/fpls.2021.759871/fullhttps://doaj.org/toc/1664-462XEach plant cell has hundreds of copies of the chloroplast genome and chloroplast transgenes do not undergo silencing. Therefore, chloroplast transformation has many powerful potential agricultural and industrial applications. We previously succeeded in integrating exogenous genes into the chloroplast genome using peptide–DNA complexes composed of plasmid DNA and a fusion peptide consisting of a cell-penetrating peptide (CPP) and a chloroplast transit peptide (cpPD complex). However, how cpPD complexes are transported into the chloroplast from outside the cell remains unclear. Here, to characterize the route by which these cpPD complexes move into chloroplasts, we tracked their movement from the extracellular space to the chloroplast stroma using a fluorescent label and confocal laser scanning microscopy (CLSM). Upon infiltration of cpPD complexes into the extracellular space of Arabidopsis thaliana leaves, the complexes reached the chloroplast surface within 6h. The cpPD complexes reached were engulfed by the chloroplast outer envelope membrane and gradually integrated into the chloroplast. We detected several cpPD complexes localized around chloroplast nucleoids and observed the release of DNA from the cpPD. Our results thus define the route taken by the cpPD complexes for gene delivery from the extracellular space to the chloroplast stroma.Kazusato OikawaAyaka TateishiMasaki OdaharaYutaka KodamaYutaka KodamaKeiji NumataKeiji NumataFrontiers Media S.A.articleArabidopsis thalianacell-penetrating peptideDNA deliverychloroplast nucleoidfield emission-scanning electron microscopeconfocal laser scanning microscopyPlant cultureSB1-1110ENFrontiers in Plant Science, Vol 12 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	Arabidopsis thaliana cell-penetrating peptide DNA delivery chloroplast nucleoid field emission-scanning electron microscope confocal laser scanning microscopy Plant culture SB1-1110
spellingShingle	Arabidopsis thaliana cell-penetrating peptide DNA delivery chloroplast nucleoid field emission-scanning electron microscope confocal laser scanning microscopy Plant culture SB1-1110 Kazusato Oikawa Ayaka Tateishi Masaki Odahara Yutaka Kodama Yutaka Kodama Keiji Numata Keiji Numata Imaging of the Entry Pathway of a Cell-Penetrating Peptide–DNA Complex From the Extracellular Space to Chloroplast Nucleoids Across Multiple Membranes in Arabidopsis Leaves
description	Each plant cell has hundreds of copies of the chloroplast genome and chloroplast transgenes do not undergo silencing. Therefore, chloroplast transformation has many powerful potential agricultural and industrial applications. We previously succeeded in integrating exogenous genes into the chloroplast genome using peptide–DNA complexes composed of plasmid DNA and a fusion peptide consisting of a cell-penetrating peptide (CPP) and a chloroplast transit peptide (cpPD complex). However, how cpPD complexes are transported into the chloroplast from outside the cell remains unclear. Here, to characterize the route by which these cpPD complexes move into chloroplasts, we tracked their movement from the extracellular space to the chloroplast stroma using a fluorescent label and confocal laser scanning microscopy (CLSM). Upon infiltration of cpPD complexes into the extracellular space of Arabidopsis thaliana leaves, the complexes reached the chloroplast surface within 6h. The cpPD complexes reached were engulfed by the chloroplast outer envelope membrane and gradually integrated into the chloroplast. We detected several cpPD complexes localized around chloroplast nucleoids and observed the release of DNA from the cpPD. Our results thus define the route taken by the cpPD complexes for gene delivery from the extracellular space to the chloroplast stroma.
format	article
author	Kazusato Oikawa Ayaka Tateishi Masaki Odahara Yutaka Kodama Yutaka Kodama Keiji Numata Keiji Numata
author_facet	Kazusato Oikawa Ayaka Tateishi Masaki Odahara Yutaka Kodama Yutaka Kodama Keiji Numata Keiji Numata
author_sort	Kazusato Oikawa
title	Imaging of the Entry Pathway of a Cell-Penetrating Peptide–DNA Complex From the Extracellular Space to Chloroplast Nucleoids Across Multiple Membranes in Arabidopsis Leaves
title_short	Imaging of the Entry Pathway of a Cell-Penetrating Peptide–DNA Complex From the Extracellular Space to Chloroplast Nucleoids Across Multiple Membranes in Arabidopsis Leaves
title_full	Imaging of the Entry Pathway of a Cell-Penetrating Peptide–DNA Complex From the Extracellular Space to Chloroplast Nucleoids Across Multiple Membranes in Arabidopsis Leaves
title_fullStr	Imaging of the Entry Pathway of a Cell-Penetrating Peptide–DNA Complex From the Extracellular Space to Chloroplast Nucleoids Across Multiple Membranes in Arabidopsis Leaves
title_full_unstemmed	Imaging of the Entry Pathway of a Cell-Penetrating Peptide–DNA Complex From the Extracellular Space to Chloroplast Nucleoids Across Multiple Membranes in Arabidopsis Leaves
title_sort	imaging of the entry pathway of a cell-penetrating peptide–dna complex from the extracellular space to chloroplast nucleoids across multiple membranes in arabidopsis leaves
publisher	Frontiers Media S.A.
publishDate	2021
url	https://doaj.org/article/c91ced86e1cc4e60ad3cff00e7db8b45
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Imaging of the Entry Pathway of a Cell-Penetrating Peptide–DNA Complex From the Extracellular Space to Chloroplast Nucleoids Across Multiple Membranes in Arabidopsis Leaves

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