Suspension protoplasts as useful experimental tool to study localization of GFP-tagged proteins in Arabidopsis thaliana

Gábor Rigó, F. Ayaydin, L. Szabados, Csaba Koncz, Ágnes Cséplo

Research output: Contribution to journalArticle

3 Citations (Scopus)

Abstract

A routinely used protocol is described here including protoplast isolation and PEG-mediated plasmid DNA transformation followed by LSM analysis. We have isolated protoplasts from suspension cultures of wild-type Arabidopsis thaliana Col-0, than protoplasts were transformed with different constructs of AtCRK5 gene tagged with GFP. The protoplast isolation and PEG-mediated transformation process took 6-8 hours. Localization studies could be carried out by LSM microscopy in 0-24 hrs followed transformation. Using this method, we could have localized both 35S::cCRK5-GFP and g::gCRK5-GFP fusion proteins in plasma membrane of cell suspension originated protoplasts, while an N-terminal myristoylation site masked version of 35S::cCRK5-GFP and the N-terminal GFP tagged 35S::GFP-cCRK5 fusion proteins could be found in cell nuclei. Mislocalization of the two last fusion proteins is in good agreement with the fact that the N-terminal myristoylation sites of these proteins were impaired. As a conclusion, the Arabidopsis suspension derived protoplast system is very quick tool for identification of protein localization and to provide possibility to obtain preliminarily information on possible protein localization prior to plant regeneration.

Original languageEnglish
Pages (from-to)59-61
Number of pages3
JournalActa Biologica Szegediensis
Volume52
Issue number1
Publication statusPublished - 2008

Fingerprint

Arabidopsis Proteins
Protoplasts
protoplasts
Suspensions
Arabidopsis thaliana
Proteins
Fusion reactions
proteins
Arabidopsis
Polyethylene glycols
cell nucleus
Cell membranes
Plasma Cells
Cell Nucleus
cell suspension culture
Regeneration
Microscopy
microscopy
plasmids
Microscopic examination

Keywords

  • Arabidopsis thaliana Col-0
  • AtCRK5
  • Fluorescence microscopy
  • PEG mediated transformation
  • Protein localization
  • Suspension protoplasts

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)
  • Agricultural and Biological Sciences(all)

Cite this

Suspension protoplasts as useful experimental tool to study localization of GFP-tagged proteins in Arabidopsis thaliana. / Rigó, Gábor; Ayaydin, F.; Szabados, L.; Koncz, Csaba; Cséplo, Ágnes.

In: Acta Biologica Szegediensis, Vol. 52, No. 1, 2008, p. 59-61.

Research output: Contribution to journalArticle

@article{7ea94883f3a042d582bb198b37f95f4f,
title = "Suspension protoplasts as useful experimental tool to study localization of GFP-tagged proteins in Arabidopsis thaliana",
abstract = "A routinely used protocol is described here including protoplast isolation and PEG-mediated plasmid DNA transformation followed by LSM analysis. We have isolated protoplasts from suspension cultures of wild-type Arabidopsis thaliana Col-0, than protoplasts were transformed with different constructs of AtCRK5 gene tagged with GFP. The protoplast isolation and PEG-mediated transformation process took 6-8 hours. Localization studies could be carried out by LSM microscopy in 0-24 hrs followed transformation. Using this method, we could have localized both 35S::cCRK5-GFP and g::gCRK5-GFP fusion proteins in plasma membrane of cell suspension originated protoplasts, while an N-terminal myristoylation site masked version of 35S::cCRK5-GFP and the N-terminal GFP tagged 35S::GFP-cCRK5 fusion proteins could be found in cell nuclei. Mislocalization of the two last fusion proteins is in good agreement with the fact that the N-terminal myristoylation sites of these proteins were impaired. As a conclusion, the Arabidopsis suspension derived protoplast system is very quick tool for identification of protein localization and to provide possibility to obtain preliminarily information on possible protein localization prior to plant regeneration.",
keywords = "Arabidopsis thaliana Col-0, AtCRK5, Fluorescence microscopy, PEG mediated transformation, Protein localization, Suspension protoplasts",
author = "G{\'a}bor Rig{\'o} and F. Ayaydin and L. Szabados and Csaba Koncz and {\'A}gnes Cs{\'e}plo",
year = "2008",
language = "English",
volume = "52",
pages = "59--61",
journal = "Acta Biologica Szegediensis",
issn = "1588-385X",
publisher = "University of Szeged",
number = "1",

}

TY - JOUR

T1 - Suspension protoplasts as useful experimental tool to study localization of GFP-tagged proteins in Arabidopsis thaliana

AU - Rigó, Gábor

AU - Ayaydin, F.

AU - Szabados, L.

AU - Koncz, Csaba

AU - Cséplo, Ágnes

PY - 2008

Y1 - 2008

N2 - A routinely used protocol is described here including protoplast isolation and PEG-mediated plasmid DNA transformation followed by LSM analysis. We have isolated protoplasts from suspension cultures of wild-type Arabidopsis thaliana Col-0, than protoplasts were transformed with different constructs of AtCRK5 gene tagged with GFP. The protoplast isolation and PEG-mediated transformation process took 6-8 hours. Localization studies could be carried out by LSM microscopy in 0-24 hrs followed transformation. Using this method, we could have localized both 35S::cCRK5-GFP and g::gCRK5-GFP fusion proteins in plasma membrane of cell suspension originated protoplasts, while an N-terminal myristoylation site masked version of 35S::cCRK5-GFP and the N-terminal GFP tagged 35S::GFP-cCRK5 fusion proteins could be found in cell nuclei. Mislocalization of the two last fusion proteins is in good agreement with the fact that the N-terminal myristoylation sites of these proteins were impaired. As a conclusion, the Arabidopsis suspension derived protoplast system is very quick tool for identification of protein localization and to provide possibility to obtain preliminarily information on possible protein localization prior to plant regeneration.

AB - A routinely used protocol is described here including protoplast isolation and PEG-mediated plasmid DNA transformation followed by LSM analysis. We have isolated protoplasts from suspension cultures of wild-type Arabidopsis thaliana Col-0, than protoplasts were transformed with different constructs of AtCRK5 gene tagged with GFP. The protoplast isolation and PEG-mediated transformation process took 6-8 hours. Localization studies could be carried out by LSM microscopy in 0-24 hrs followed transformation. Using this method, we could have localized both 35S::cCRK5-GFP and g::gCRK5-GFP fusion proteins in plasma membrane of cell suspension originated protoplasts, while an N-terminal myristoylation site masked version of 35S::cCRK5-GFP and the N-terminal GFP tagged 35S::GFP-cCRK5 fusion proteins could be found in cell nuclei. Mislocalization of the two last fusion proteins is in good agreement with the fact that the N-terminal myristoylation sites of these proteins were impaired. As a conclusion, the Arabidopsis suspension derived protoplast system is very quick tool for identification of protein localization and to provide possibility to obtain preliminarily information on possible protein localization prior to plant regeneration.

KW - Arabidopsis thaliana Col-0

KW - AtCRK5

KW - Fluorescence microscopy

KW - PEG mediated transformation

KW - Protein localization

KW - Suspension protoplasts

UR - http://www.scopus.com/inward/record.url?scp=59949097576&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=59949097576&partnerID=8YFLogxK

M3 - Article

AN - SCOPUS:59949097576

VL - 52

SP - 59

EP - 61

JO - Acta Biologica Szegediensis

JF - Acta Biologica Szegediensis

SN - 1588-385X

IS - 1

ER -