106
H. Pfanz
5.2.3 Techniques to Determine Intra- and Intercellular pH
There are several methods available for the determination of inter- and
intracellular pH values in animal and plant cells. As there is a vast amount
of literature on these techniques (for recent reviews, see Kurkdjian and
Guern 1989a; Pfanz and Heber 1989), only the basic principles will be
summarized here.
To estimate the extracellular proton concentration of root cells, microelectrodes localized only a few microns from root surfaces have been used
(Newman et al. 1987). Generally, the classical, but still valuable method of
using pH-indicating dyes in agar (Marschner et al. 1982) is used for estimating extracellular pH in roots of intact plants, whereas for leaves, flat pH
electrodes are pressed to partially abraded epidermal tissue (Lursen 1978).
The magnitude and the changes of fluxes of weak acids across the plasmalemma are also used for extracellular pH estimations (Grignon and
Sentenac 1991). Fluorescent dyes infiltrated into leaves allow qualitative and
quantitative estimations of extracellular pH (Pfanz and Dietz 1987; Canny
1988; Edwards et al. 1988). Other attempts use fluids extracted via centrifugation (Madore and Webb 1981)· or apply pressure to extract an extracellular sap (Hartung 'et al. 1988). A recent approach using fluorescence
was described by Hoffmann et al. in 1992.
The spectrum of methods for measuring symplastic pH values is even
broader than that described for the cell wall phase (see Caldwell 1956;
Boron and Roos 1976; Kurkdjian and Guern 1989a; Pfanz and Heber 1989).
A crude, but quick and easy method to use for all plant materials is pH
measurement in homogenized extracts of plant organs, of isolated cells or
protoplasts, or even of isolated organelles (Clevenger 1919; Pfanz 1987;
Wagner 1990; Pfanz and Beyschlag 1992). The main limitation is that with
this crude method mainly vacuolar pH is measured.
H+ -selective microelectrodes have extensively been used to study cytoplasmic and vacuolar pH values (Felle and Bertl 1986; Steigner et al. 1988).
Chemical probes also found broad application. This technique is based on
the different permeation characteristics of undissociated and dissociated
probes. Generally, fluorescent or radiolabeled compounds have been used
as probes in such procedures (for details see Kurkdjian and Guern 1989a;
Pfanz and Heber 1989 and references therein). One of the most widely used
compounds in this context is 14C-DMO (5.5-dimethyl-oxazolidine-2.4-dione;
Waddell and Butler 1959; Pfanz et al. 1987; Kurkdjian and Guern 1989b).
Recently, pyranine and carboxyfluoresceine have been used to follow the
kinetics of intracellular pH changes in C3 plants during light/dark transitions
and during exposure to potentially acidic air pollutants (Yin et al. 1990,
1991).
Furthermore, gas exchange with very fast IRGA systems has been applied to detect chloroplastic pH values (Oja et al. 1986). A very intriguing,
noninvasive method for pH estimations in (nearly) intact leaf tissues is 31p
H. Pfanz
5.2.3 Techniques to Determine Intra- and Intercellular pH
There are several methods available for the determination of inter- and
intracellular pH values in animal and plant cells. As there is a vast amount
of literature on these techniques (for recent reviews, see Kurkdjian and
Guern 1989a; Pfanz and Heber 1989), only the basic principles will be
summarized here.
To estimate the extracellular proton concentration of root cells, microelectrodes localized only a few microns from root surfaces have been used
(Newman et al. 1987). Generally, the classical, but still valuable method of
using pH-indicating dyes in agar (Marschner et al. 1982) is used for estimating extracellular pH in roots of intact plants, whereas for leaves, flat pH
electrodes are pressed to partially abraded epidermal tissue (Lursen 1978).
The magnitude and the changes of fluxes of weak acids across the plasmalemma are also used for extracellular pH estimations (Grignon and
Sentenac 1991). Fluorescent dyes infiltrated into leaves allow qualitative and
quantitative estimations of extracellular pH (Pfanz and Dietz 1987; Canny
1988; Edwards et al. 1988). Other attempts use fluids extracted via centrifugation (Madore and Webb 1981)· or apply pressure to extract an extracellular sap (Hartung 'et al. 1988). A recent approach using fluorescence
was described by Hoffmann et al. in 1992.
The spectrum of methods for measuring symplastic pH values is even
broader than that described for the cell wall phase (see Caldwell 1956;
Boron and Roos 1976; Kurkdjian and Guern 1989a; Pfanz and Heber 1989).
A crude, but quick and easy method to use for all plant materials is pH
measurement in homogenized extracts of plant organs, of isolated cells or
protoplasts, or even of isolated organelles (Clevenger 1919; Pfanz 1987;
Wagner 1990; Pfanz and Beyschlag 1992). The main limitation is that with
this crude method mainly vacuolar pH is measured.
H+ -selective microelectrodes have extensively been used to study cytoplasmic and vacuolar pH values (Felle and Bertl 1986; Steigner et al. 1988).
Chemical probes also found broad application. This technique is based on
the different permeation characteristics of undissociated and dissociated
probes. Generally, fluorescent or radiolabeled compounds have been used
as probes in such procedures (for details see Kurkdjian and Guern 1989a;
Pfanz and Heber 1989 and references therein). One of the most widely used
compounds in this context is 14C-DMO (5.5-dimethyl-oxazolidine-2.4-dione;
Waddell and Butler 1959; Pfanz et al. 1987; Kurkdjian and Guern 1989b).
Recently, pyranine and carboxyfluoresceine have been used to follow the
kinetics of intracellular pH changes in C3 plants during light/dark transitions
and during exposure to potentially acidic air pollutants (Yin et al. 1990,
1991).
Furthermore, gas exchange with very fast IRGA systems has been applied to detect chloroplastic pH values (Oja et al. 1986). A very intriguing,
noninvasive method for pH estimations in (nearly) intact leaf tissues is 31p