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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

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: Oikawa, Kazusato, Tateishi, Ayaka, Odahara, Masaki, Kodama, Yutaka, Numata, Keiji
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Frontiers Media S.A. 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8678410/
https://www.ncbi.nlm.nih.gov/pubmed/34925409
http://dx.doi.org/10.3389/fpls.2021.759871
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author Oikawa, Kazusato
Tateishi, Ayaka
Odahara, Masaki
Kodama, Yutaka
Numata, Keiji
author_facet Oikawa, Kazusato
Tateishi, Ayaka
Odahara, Masaki
Kodama, Yutaka
Numata, Keiji
author_sort Oikawa, Kazusato
collection PubMed
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.
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spelling pubmed-86784102021-12-18 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 Oikawa, Kazusato Tateishi, Ayaka Odahara, Masaki Kodama, Yutaka Numata, Keiji Front Plant Sci Plant Science 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. Frontiers Media S.A. 2021-12-03 /pmc/articles/PMC8678410/ /pubmed/34925409 http://dx.doi.org/10.3389/fpls.2021.759871 Text en Copyright © 2021 Oikawa, Tateishi, Odahara, Kodama and Numata. https://creativecommons.org/licenses/by/4.0/This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
spellingShingle Plant Science
Oikawa, Kazusato
Tateishi, Ayaka
Odahara, Masaki
Kodama, Yutaka
Numata, Keiji
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 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_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_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
topic Plant Science
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8678410/
https://www.ncbi.nlm.nih.gov/pubmed/34925409
http://dx.doi.org/10.3389/fpls.2021.759871
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