Loading acetoxymethyl ester fluorescent dyes into the cytoplasm of Arabidopsis and Commelina guard cells

Kazuyuki Kuchitsu, John M. Ward, Gethyn J. Allen, Ilona Schelle, Julian I. Schroeder

Research output: Contribution to journalArticle

18 Citations (Scopus)

Abstract

Cytosolic calcium and pH changes are integral components of guard cell signal transduction. Acetoxymethyl (AM) ester-linked Ca2+ sensitive dyes are usually degraded before loading by extracellular esterases or partitioned into plant vacuoles using standard techniques, thereby preventing cytoplasmic Ca2+ imaging. Here a method is described for improved loading of the calcium sensitive fluorescent dyes Calcium Green-1 and Fura-2 AM ester into the cytoplasm of Commelina and Arabidopsis guard cells in epidermal strips, allowing fluorescence from several guard cells in an epidermal strip to be imaged and measured simultaneously. Calcium Green-1 based imaging, external Ca2+ buffering and Mn2+ quenching in Commelina guard cells suggest that abscisic acid stimulates plasma membrane Ca2+ influx. The Ca2+ sensitive dye Fura-2 was loaded into the cytoplasm of Arabidopsis guard cells, allowing ratiometric analyses of these cells. Data indicated that intact Ca2+ homeostasis mechanisms were present in Fura-2 AM loaded cells. Loading of acetoxymethyl ester dyes provides a viable alternative method to cameleon imaging, which will be useful in loading Arabidposis mutants that are difficult to transform. This method may also be applicable to loading other ester linked dyes into the cytoplasm of plant cells.

Original languageEnglish (US)
Pages (from-to)527-533
Number of pages7
JournalNew Phytologist
Volume153
Issue number3
DOIs
StatePublished - Jan 1 2002

Fingerprint

Commelina
fluorescent dyes
guard cells
Fluorescent Dyes
Arabidopsis
Cytoplasm
Esters
cytoplasm
esters
calcium
Coloring Agents
Fura-2
dyes
Calcium
image analysis
Abscisic Acid
Plant Cells
Esterases
Cellular Structures
Vacuoles

Keywords

  • Abscisic acid (ABA)
  • Acetoxymethyl ester fluorescent dye
  • Arabidopsis
  • Calcium Green-1, Fura-2
  • Calcium imaging
  • Commelina
  • Guard cell

Cite this

Loading acetoxymethyl ester fluorescent dyes into the cytoplasm of Arabidopsis and Commelina guard cells. / Kuchitsu, Kazuyuki; Ward, John M.; Allen, Gethyn J.; Schelle, Ilona; Schroeder, Julian I.

In: New Phytologist, Vol. 153, No. 3, 01.01.2002, p. 527-533.

Research output: Contribution to journalArticle

Kuchitsu, Kazuyuki ; Ward, John M. ; Allen, Gethyn J. ; Schelle, Ilona ; Schroeder, Julian I. / Loading acetoxymethyl ester fluorescent dyes into the cytoplasm of Arabidopsis and Commelina guard cells. In: New Phytologist. 2002 ; Vol. 153, No. 3. pp. 527-533.
@article{8682a9fced734222affc179c65baf3c9,
title = "Loading acetoxymethyl ester fluorescent dyes into the cytoplasm of Arabidopsis and Commelina guard cells",
abstract = "Cytosolic calcium and pH changes are integral components of guard cell signal transduction. Acetoxymethyl (AM) ester-linked Ca2+ sensitive dyes are usually degraded before loading by extracellular esterases or partitioned into plant vacuoles using standard techniques, thereby preventing cytoplasmic Ca2+ imaging. Here a method is described for improved loading of the calcium sensitive fluorescent dyes Calcium Green-1 and Fura-2 AM ester into the cytoplasm of Commelina and Arabidopsis guard cells in epidermal strips, allowing fluorescence from several guard cells in an epidermal strip to be imaged and measured simultaneously. Calcium Green-1 based imaging, external Ca2+ buffering and Mn2+ quenching in Commelina guard cells suggest that abscisic acid stimulates plasma membrane Ca2+ influx. The Ca2+ sensitive dye Fura-2 was loaded into the cytoplasm of Arabidopsis guard cells, allowing ratiometric analyses of these cells. Data indicated that intact Ca2+ homeostasis mechanisms were present in Fura-2 AM loaded cells. Loading of acetoxymethyl ester dyes provides a viable alternative method to cameleon imaging, which will be useful in loading Arabidposis mutants that are difficult to transform. This method may also be applicable to loading other ester linked dyes into the cytoplasm of plant cells.",
keywords = "Abscisic acid (ABA), Acetoxymethyl ester fluorescent dye, Arabidopsis, Calcium Green-1, Fura-2, Calcium imaging, Commelina, Guard cell",
author = "Kazuyuki Kuchitsu and Ward, {John M.} and Allen, {Gethyn J.} and Ilona Schelle and Schroeder, {Julian I.}",
year = "2002",
month = "1",
day = "1",
doi = "10.1046/j.0028-646X.2001.00346.x",
language = "English (US)",
volume = "153",
pages = "527--533",
journal = "New Phytologist",
issn = "0028-646X",
publisher = "Wiley-Blackwell",
number = "3",

}

TY - JOUR

T1 - Loading acetoxymethyl ester fluorescent dyes into the cytoplasm of Arabidopsis and Commelina guard cells

AU - Kuchitsu, Kazuyuki

AU - Ward, John M.

AU - Allen, Gethyn J.

AU - Schelle, Ilona

AU - Schroeder, Julian I.

PY - 2002/1/1

Y1 - 2002/1/1

N2 - Cytosolic calcium and pH changes are integral components of guard cell signal transduction. Acetoxymethyl (AM) ester-linked Ca2+ sensitive dyes are usually degraded before loading by extracellular esterases or partitioned into plant vacuoles using standard techniques, thereby preventing cytoplasmic Ca2+ imaging. Here a method is described for improved loading of the calcium sensitive fluorescent dyes Calcium Green-1 and Fura-2 AM ester into the cytoplasm of Commelina and Arabidopsis guard cells in epidermal strips, allowing fluorescence from several guard cells in an epidermal strip to be imaged and measured simultaneously. Calcium Green-1 based imaging, external Ca2+ buffering and Mn2+ quenching in Commelina guard cells suggest that abscisic acid stimulates plasma membrane Ca2+ influx. The Ca2+ sensitive dye Fura-2 was loaded into the cytoplasm of Arabidopsis guard cells, allowing ratiometric analyses of these cells. Data indicated that intact Ca2+ homeostasis mechanisms were present in Fura-2 AM loaded cells. Loading of acetoxymethyl ester dyes provides a viable alternative method to cameleon imaging, which will be useful in loading Arabidposis mutants that are difficult to transform. This method may also be applicable to loading other ester linked dyes into the cytoplasm of plant cells.

AB - Cytosolic calcium and pH changes are integral components of guard cell signal transduction. Acetoxymethyl (AM) ester-linked Ca2+ sensitive dyes are usually degraded before loading by extracellular esterases or partitioned into plant vacuoles using standard techniques, thereby preventing cytoplasmic Ca2+ imaging. Here a method is described for improved loading of the calcium sensitive fluorescent dyes Calcium Green-1 and Fura-2 AM ester into the cytoplasm of Commelina and Arabidopsis guard cells in epidermal strips, allowing fluorescence from several guard cells in an epidermal strip to be imaged and measured simultaneously. Calcium Green-1 based imaging, external Ca2+ buffering and Mn2+ quenching in Commelina guard cells suggest that abscisic acid stimulates plasma membrane Ca2+ influx. The Ca2+ sensitive dye Fura-2 was loaded into the cytoplasm of Arabidopsis guard cells, allowing ratiometric analyses of these cells. Data indicated that intact Ca2+ homeostasis mechanisms were present in Fura-2 AM loaded cells. Loading of acetoxymethyl ester dyes provides a viable alternative method to cameleon imaging, which will be useful in loading Arabidposis mutants that are difficult to transform. This method may also be applicable to loading other ester linked dyes into the cytoplasm of plant cells.

KW - Abscisic acid (ABA)

KW - Acetoxymethyl ester fluorescent dye

KW - Arabidopsis

KW - Calcium Green-1, Fura-2

KW - Calcium imaging

KW - Commelina

KW - Guard cell

UR - http://www.scopus.com/inward/record.url?scp=0036186346&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0036186346&partnerID=8YFLogxK

U2 - 10.1046/j.0028-646X.2001.00346.x

DO - 10.1046/j.0028-646X.2001.00346.x

M3 - Article

AN - SCOPUS:0036186346

VL - 153

SP - 527

EP - 533

JO - New Phytologist

JF - New Phytologist

SN - 0028-646X

IS - 3

ER -