Journal of Experimental Botany, Vol. 49, No. 323, pp. 987–995, June 1998
The apoplastic pH of the Zea mays root cortex as
measured with pH-sensitive microelectrodes:
aspects of regulation
Hubert H. Felle1
Botanisches Institut I, Justus-Liebig-Universität Giessen, Senckenbergstr. 17, D–35390 Giessen, Germany
Received 19 November 1997; Accepted 2 February 1998
Abstract
Introduction
In the root cortex of Zea mays the apoplastic pH and
aspects of its regulation were investigated using pHsensitive microelectrodes. To measure the pH directly
in different cell layers of the apoplast sharp doublebarrelled electrodes were applied, whereas blunt pHelectrodes were used simultaneously to measure the
pH at the root surface. Recordings carried out
8–10 mm behind the root tip show that the apoplastic
pH is maintained between 5.1 and 5.6, depending on
the given experimental conditions, i.e. varying external
[K+], [Ca2+], pH, weak buffering, as well as perfusion
of the test medium. When the medium pH (bulk) differs
considerably from the apoplastic pH, a small pH gradient is built up between the root surface (unstirred
layer) and the outer cortex layers. In a standing
medium these gradients equilibrate. The apoplastic pH
responds to increases in external [K+] and [Ca2+] with
an acidification, which is attributed to ion-exchange
properties of the cell wall constituents. Stimulation of
proton pump activity with fusicoccin acidifies the apoplast from pH 5.6 to pH 4.8, while deactivation of the
pump with cyanide/salicylhydroxamic acid increases
the pH of the apoplast from 5.6 to 6.2, and further to
pH 6.6 with CCCP. The Ca2+ channel antagonists
nifedipine and La3+ also increase the apoplastic pH.
It is suggested that not only the proton pump, but also
the cation channels may contribute to the regulation
of the apoplastic pH.
The structural and ionic characteristics of the apoplast of
higher plants are important because (a) they determine
the ionic composition of the medium adjacent to the
plasma membrane, (b) they control the transport of
solutes, and (c) they affect mechanical and osmotic phenomena involved in cell growth (Grignon and Sentenac,
1991). The nutrition of a plant or a plant cell depends
on its ability to recognize, bind and transport a variety
of substances, which cross the root cortex either symplastically, transcellularly or apoplastically. The mode of transport depends very much on the physico-chemical
properties of the apoplast, and on the form and concentration of the substance which is to be taken up into the
symplast. The constituents of the apoplast of a primary
root are freely exchangeable with the rhizosphere.
However, dynamics of this process are not merely passive,
since the apoplast is surrounded by plasma membrane
which, through its transport activity, will markedly influence the milieu of the apoplast. Since, in plants, many of
the transport processes across the plasma membrane
depend directly or indirectly on proton transport or
proton turnover, the pH of the root apoplast and the
dynamics of this is of major importance for the transport
of organic substrates and ions in roots.
For this report, the apoplastic pH of the root cortex
has been measured directly using pH-sensitive microelectrodes. In order to make a contribution to our general
understanding of the regulation of the apoplastic pH and
its dynamics, the ionic conditions in the apoplast and in
the regions next to it were manipulated, while the pH of
the apoplast and of the root surface was recorded continuously and simultaneously. It is demonstrated that the pH
Key words: Apoplast, ion-selective microelectrodes, pH,
unstirred layer, Zea mays, root.
1 Fax: +49 641 99 35119. E-mail: [email protected]
© Oxford University Press 1998
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Felle
of the root cortex apoplast of Zea mays is maintained in
the range 5.1 to 5.6 through an interaction between ion
exchange, proton transport and channel activity.
Materials and methods
General conditions
Seeds of Zea mays L. (helix) were first soaked for 12 h in tap
water, placed on moist filter paper for approximately 2 d, and
then transferred on to a Plexiglas grid through which the roots
grew into a constantly aerated solution (1 mol m−3 KNO ,
3
NaCl, CaCl , each) without developing root hairs. Only intact
2
5-d-old seedlings were used for the experiments. The seedlings
were placed into a cuvette which consisted of two chambers,
one of which held the shoot with endosperm, and the other was
designed to hold the primary root and permitted a constant
perfusion (usually 1–2 ml min−1). This arrangement served to
keep the entire plant moist for the duration of the experiment
and permitted the independent horizontal approach of electrodes
from opposite sides. Prior to tests the root was kept for
approximately 1 h at rest in the chamber. As soon as the root
had resumed proper growth (~2 mm h−1), the electrodes were
brought into position. Measurements were carried out in a
weakly buffered solution which consisted of 2-[N-morpholino]ethanesulphuric acid (MES) and tris(hydroxymethyl )-aminomethane ( TRIS) (0.5 mol m−3, each), mixed to the desired pH
and supplemented with KCl, NaCl, CaCl , and agents, as given
2
in the legends. CCCP (carbonylcyanide–3-chlorophenylhydrazone) was added from a 20 mol m−3 ethanolic stock solution.
To determine the time a change in solution takes, after
each experimental series the response of the surface pHelectrode was tested through a pH-jump. Thus, the lines in the
graphs indicate the instant the respective test solution reaches
the root.
Fabrication of the pH-sensitive microelectrodes for extracellular
use
The electrical set-up for the impalement of root hairs, the
fabrication and application of ion-sensitive microelectrodes and
their intracellular application has been described (Felle, 1987,
1994; Felle and Bertl, 1986). The fabrication of the ion-selective
electrodes for extracellular use differed in that the tip was
approximately 5 mm in diameter, blunt and heat-polished. To
give the sensor in the tip sufficient firmness to stay in place for
repeated use, the cocktail (Fluka) was dissolved in a mixture
of 40 mg polyvinylchloride (PVC ) per ml of tetrahydrofuran
( THF ) at a ratio of 30/70 (v/v). After evaporation of the THF,
the remaining firm gel was topped with the undiluted sensor
cocktail, followed by the reference solution. After equilibrating,
these electrodes gave stable responses for at least 2 weeks, when
stored in a dry chamber. During measurements, the electrodes
were connected to a high-impedance amplifier (FD 223; WPInstruments, Sarasota, Fla, USA) which simultaneously measured the signals coming from the ion-selective electrode and the
voltage reference. Signals were recorded on a chart recorder (L
2200, Linseis, Germany).
Measurements in the apoplast
For measurements in the apoplast double-barrelled microelectrodes were fabricated from ‘piggy-back’ rods ( WPIInstruments, Sarasota, Fla, USA; Hilgenberg, Malsdorf,
Germany). The main barrel was used as the ion-sensitive
electrode and was filled as described above. To compensate for
differences in (turgor-) pressure while driving the electrode in
and out of cells, electrodes were connected to a home-built
pressure controller (Herrmann and Felle, 1995). The small
barrel was filled with 500 mol m−3 KCl and represented a
voltage reference. In order to perform an apoplastic
measurement, these electrodes were carefully inserted into the
Zea mays root, 8–10 mm behind the tip. Care was taken that
the first impalement resulted in a clean intracellular pHmeasurement (Fig. 1). To test the proper function of the
electrode, mild standard tests (e.g. changing external pH ) were
carried out routinely. Pushing the electrode further into the
root cortex resulted in immediate loss of the intracellular
recording and, provided the injury was not severe, usually was
followed by a second intracellular measurement in the adjacent
cell. This procedure was continued until the tip of the electrode
hit a radial cell wall which gave entirely different signals on
both electrodes. In that case the pH-signal dropped from
slightly alkaline (cytosol ) to acidic values below 6, while the
voltage dropped to −30 to −40 mV. The latter indicated that
the electrode was not placed within the similar acidic vacuole,
but represented an apoplastic recording. As soon as the pHelectrode measured clearly within the apoplast, a reproducible
pH was recorded, which depended, however, on a variety of
conditions (see below). Recordings which had drifts or were
not stable on either of the electrodes were not continued. The
success rate of the described procedure was about 10%. This
appears low, but as soon as the electrode was in place,
experiments could be performed for hours. To obtain
continuous readings of the medium pH during the change
in conditions, a second pH-electrode displaying the same
calibration slope as the apoplastic electrode, was placed in
the bulk of the medium. Measurements of apoplastic K+concentrations were carried out in the same manner as the
pH-measurements.
Fig. 1. Protocol of a pH measurement within the root apoplast. A
double-barrelled pH/voltage-microelectrode is inserted into the first
cortex cell of a Zea mays root, i.e. cytosolic pH (pH ) and membrane
potential (E ) are measured simultaneously. Moving the electrode
m
further into the second or third cell layer (2.C, 3.C ) results in loss of
the intracellular recording (spikes). Pushing the electrode deeper into
the cortex either leads to more intracellular recordings, or a radial cell
wall is hit which results in a drop in voltage and pH. Once this
recording becomes stable, it is accepted as an apoplastic recording. P.
on/off=perfusion on/off. See text.
Apoplastic pH 989
Results
pH-relationships of medium, root surface and apoplast
The fine water film covering the root surface mediates
between the ionic conditions of the rhizosphere and the
apoplast. In vitro, this film is represented by the so-called
‘unstirred layer’, the dimension of which depends critically
on the velocity of perfusion within the test chamber. Since
the plasma membrane as well as the apoplast is in direct
contact with the most inner parts of the unstirred layer,
it is important to know the composition, the dimension
and the dynamics of this layer under the given experimental conditions. When a pH-sensitive microelectrode
is moved at an angle of approximately 90 degrees from
the bulk of the medium towards the root tip and then
along the root at a constant distance from it, a characteristic pH-profile is measured ( Fig. 2a), which looks very
much like the pH pattern reported by Pilet et al. (1983).
A sharp acidic peak at the meristematic zone is followed
by a less acidic zone at around 2 mm. Moving the
electrode further along the root, the pH then decreases
again to obtain a relatively stable value behind the
elongation zone. Although such a pH-profile may be
physiologically meaningful for root growth, this aspect
will be dealt with elsewhere (Peters and Felle, unpublished
observations). For apoplastic measurements the zone of
the least pH fluctuations, i.e. 8–10 mm behind the tip,
was chosen.
Figure 2a shows that under most conditions the root
surface pH is clearly more acidic than the medium pH,
unless the latter falls below pH 6. Thus, the pH-profile
taken at a bulk pH of 4.8, displays large parts of the
profile which are less acidic than the bulk. Perfusion
(on/off ) disturbs the measured surface pH. In Fig. 2b,
the kinetics of the surface pH following a medium pH
change from 7.8 to 4.9 are shown. Clearly, the pH at the
root surface is roughly 2 units more acidic than the
medium pH of 7.8, but becomes less acidic when perfusion
is occurring, indicating a washing effect. When the
medium pH drops to 4.8, the surface pH becomes less
acidic than the bulk pH. In this case perfusion has only
minor effects on the surface pH. No differences between
surface and bulk pH are observed when the bulk pH is
5.5, indicating the pH range where apoplast and unstirred
layer meet.
The relationship between the pH of the medium, the
root surface and the cortical apoplast is shown in Fig. 3.
At a given pH 6.5 there is a relatively steep pH-gradient
reaching from the bulk of the medium towards the root
surface and into the apoplast, while the perfusion is on
( Fig. 3a). Clearly, the steepness of these gradients depends
on the local velocity of the medium passing the root
surface. In fact, when perfusion is stopped, the pHgradient becomes more and more shallow with time, and
the pH of the root surface and of the apoplast equilibrate.
Figure 3b compares the pH-gradients in the medium and
in the apoplast in the presence of different medium pHs
during perfusion. At pH 5.2 no significant differences in
pH between medium and apoplast are recorded, a finding
which corresponds to the data shown in Fig. 2b.
Fig. 2. pH-measurements at the root surface using a blunt pH-sensitive microelectrode. (a) After setting the medium pH (circled symbols) the
electrode is moved from the bulk of the medium (Bulk) towards the root tip ( Tip). Longitudinal pH-profiles are then measured by moving the pHelectrode along the root at a constant distance of 10 mm, and at different bulk pHs, as indicated by location of the different symbols. Data points
were taken at defined intervals controlled with an ocular micrometer. For better assignment of the data, the symbols of the bulk pHs have been
circled in and connected with the pH values measured at the tip. Representative of 15 equivalent experiments. (b) Convergence of the surface pH
(S) and bulk pH (B) during the response to changes in medium pH from 7.9 to 4.8 and from 6.8 to 5.5. Note that both graphs (a, b) share a
common pH-scale (external pH ). Measurements were carried out 9 mm behind the tip and are representative of seven equivalent tests.
990
Felle
Fig. 3. pH-gradients within the medium and in different layers of the cortex apoplast of Zea mays roots. (a) At a fixed medium pH of 6.5 and
under various conditions of perfusion, as indicated. (b) At different medium pHs, as indicated by location of the symbols at 1000 mm. ‘0’ marks
the boundary between root cortex and the medium. Representative of six equivalent test series.
Implications of the plasma membrane proton pump for the
apoplastic pH
The plasma membrane H+ ATPase, as the primary electrogenic proton pump drives cotransport, such that a
fraction of the extruded protons re-enter the cell. When
this dynamic equilibrium is disturbed, for example,
through a change in pump activity, a pH shift to the one
side or the other should be observed. The degree to which
this shift occurs should have some relationship with the
ability of the proton pump to influence the apoplastic pH.
It is a well-known fact that fusicoccin ( FC ) stimulates
the proton pump of higher plants, thus causing increased
proton extrusion and membrane hyperpolarization
(Marrè, 1979). Since the apoplastic space is very small,
a stimulation of proton extrusion should rapidly acidify
the apoplast. Figure 4a shows that FC indeed acidifies
the apoplast and the root surface, however, it does so
relatively slowly with a small magnitude, namely to about
pH 4.8. The simultaneously measured surface pH drops
more rapidly, but remains slightly less acidic than the
apoplast. Without FC, the surface pH remains around
pH 6 ( Fig. 4a, inset).
Inversely, when the pump is inhibited or deactivated,
apoplastic pH should increase. Here a mixture of cyanide
(to inhibit oxidative phosphorylation) and salicylhydroxamic acid (to inhibit the cyanide-insensitive bypass) was
used for this purpose. As Fig. 4b shows, the apoplastic
pH indeed becomes less acidic, but not as much as one
would have expected. Although pH starts to increase
rapidly at first, the change soon becomes much slower.
Since electrode drifts could not be excluded while measuring small pH changes over a relatively long period of
time, the experiment was interrupted after about 45 min.
When CCCP was added then, the increase in apoplastic
pH was fast and became almost completely equilibrated
Fig. 4. Proton pump acitivity and apoplastic pH of Zea mays roots.
(a) The effect of 2 mmol m−3 fusicoccin (FC ) on the pH of the apoplast
and surface with and without perfusion (P. on/off ). Inset: Kinetics of
surface pH without FC, with and without perfusion. (b) Effect of
0.5 mol m−3 NaCN (CN−) plus 0.2 mol m−3 salicylhydroxamic acid
(SHAM ), and 20 mmol m−3 CCCP on apoplastic pH. CCCP was
added after preincubation in CN−/SHAM for 45 min. W=removal of
all inhibitors. Representative of five equivalent tests, each. Confidence
limits for the final pH were: for FC 4.73 to 4.97; for CN/SHAM 6.13
to 6.29 and for CCCP 6.55 to 6.63.
Apoplastic pH 991
with the medium pH of 6.8. These data indicate that the
plasma membrane proton pump is not the only factor in
apoplastic pH regulation.
The effects of K+, Ca2+ and Cl− on apoplastic pH
The negative charges of the cell wall pectin constituents
(e.g. galacturonic acid) bind cations which, in the case of
Ca2+, will contribute considerably to the firmness of the
cell wall. Since these bonds are relatively weak, protons
can to some extent replace cations from their binding
sites and thus loosen the cell wall (Jarvis, 1982; Homblé
et al., 1989) Thus, the ratio of bound/free ions depends
on the composition of the apoplastic medium, but even
more so on the composition of the unstirred root surface
layer. This is demonstrated in Figs 5 and 6. Figure 5
compares the development of surface pH with that of the
apoplast (third to fifth cell layer) during an increase in
external KCl concentration from 0.1 to 10 mol m−3.
Whereas the pH of the surface (bulk pH 7.3) decreases
by about 0.3 units, the pH of the apoplast responds more
slowly and to a lesser extent. When perfusion is stopped,
both surface and apoplastic pH drop further. These
responses are fully reversible, albeit with different timecourses (kinetics not shown).
As Fig. 6 shows, Ca2+ is apparently less effective in
influencing apoplastic or even surface pH. This seems to
depend on the concentration of KCl in the medium (Fig.
6a). Thus, in the presence of low KCl, i.e. 0.01 to 0.1
mol−3, an acidification is observed following the increase
in external [Ca2+] from 0.1 to 10 mol m−3, however, an
alkalinization occurs at higher KCl concentrations. This
somewhat unexpected behaviour seems to depend on the
presence of chloride. Thus, when Cl− is exchanged for
the non-permeant gluconate, Ca2+ acidifies both root
surface and apoplast ( Fig. 6b).
The effect of changes in external [ K+] on the apoplastic
pH depends on the cell layer the measurements are carried
out in, and is generally lower in the inner cortex. In an
effort to explain this, the apoplastic [ K+] was measured
in the different cell layers of the root cortex, by applying
a K+-selective electrode in the same manner as the pHelectrode. Figure 7 shows that in fact there are significant
[ K+] differences between medium and root surface. There
are [ K+]-gradients within the apoplast which depend on
the K+-concentration of the medium, i.e. the apoplastic
[ K+] is higher than that of the medium at an external
[ K+] of 1 mol m−3 or less, but is lower when the
medium [ K+] is 10 mol m−3 or higher, a behaviour which
indicates regulation. There is also a temporal aspect:
approximately 1 h after setting the external [ K+],
the apoplastic [ K+] in the 6/7th cell layer reaches
from 3.3±0.9 mol m−3 (SD; n=6; external [ K+]=
0.01 mol m−3) to 18.2±5.1 mol m−3 (SD; n=6; external
[ K+]=30 mol m−3). Exposing the root for 5 h to these
concentrations reduces the differences in the apoplastic
[ K+] to 4.9±1.2 mol m−3 (SD; n=5) and 11.6±2.9 mol
m−3 (SD; n=5), respectively (data not shown). This
indicates that, although the apoplastic [ K+] is regulated,
there is a concentration gradient from the surface to the
inner cortex layers, depending on the external [ K+].
The effect of channel inhibitors on apoplastic pH
Since changes in the cation concentration of the apoplast
can obviously shift the pH of the apoplast, the activity
of their conducting channels should influence the apoplastic pH to some extent. As Fig. 8 demonstrates, this
is indeed the case. 0.1 mol m−3 nifedipine increases the
apoplastic pH by about 0.1 unit, while the same concentration of LaCl causes a much stronger, albeit partly
3
transient increase in apoplastic pH. These data indicate
that the pH of the apoplast may be influenced through
the regulation of cation conducting channels.
Discussion
Fig. 5. Influence of external [ K+] on the pH of the cortex apoplast of
Zea mays roots (upper curve) and of the root surface ( lower curve).
Prior to the addition of 10 mol m−3 KCl roots were kept for
approximately 1 h in 0.1 mol m−3 KCl. Additionally, the effect of
perfusion (off/on) was tested. Representative kinetics of 11 equivalent
experiments.
Several important points are demonstrated: (1) Ionselective microelectrodes are useful tools for the investigation of the apoplastic ionic milieu. (2) In weakly
buffered solutions ranging from pH 5 to pH 8 the maize
root cortex is able to stabilize the pH of its immediate
environment to 5.1–6.0, and of its apoplastic compartment to 5.1–5.6: (a) by controlling the activity of the
plasma membrane proton pump of its constituent cells;
(b) by controlling the activity of plasma membrane
channels transporting other ions; (c) by adjusting the
ionic composition of the apoplast by exchanging ions
with the cell wall.
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Felle
Fig. 6. Influence of external [Ca2+] on the pH of the cortex apoplast of Zea mays roots (upper curves) and of the root surface ( lower curves). (a)
Effect of 10 mol m−3 CaCl in the presence of different external [ K+] (preincubated for 1 h). Confidence limits: for the apoplast the maximal pH
2
changes were +0.18 to +0.22 ( KCl, 10 mM ), −0.07 to −0.11 ( KCl, 0.1 mM ), −0.08 to −0.13 ( KCl, 0.01 mM ). For the surface the maximal
pH changes were +0.33 to +0.37 ( KCl, 10 mM ). ‘+’=increase in pH, ‘−’=decrease in pH. (b) Effect of 10 mol m−3 CaCl (CaCl ) or Ca2
2
gluconate (Ca-Glu). Roots were preincubated in 0.1 mol m−3 CaCl (+1 mol m−3 KCl ) for approximately 1 h, and the pH kinetics of both apoplast
2
and surface were measured with and without perfusion (P.on/off ). Kinetics are representative of at least six equivalent experiments, each.
Ion-selective microelectrodes, a convenient direct probe for
the investigation of the apoplastic ion milieu
Fig. 7. [ K+]-gradients between the external medium (B), surface (‘0’)
and different layers of the cortex apoplast of Zea mays roots (numbers
on abscissa), measured with a K+-selective microelectrode. Roots were
equilibrated for approximately 1 h to the respective [ K+] in the medium,
indicated by the symbols. For clarity reasons no error margins are
given. Two points per layer and [ K+] denote the highest and lowest
concentration measured. See text.
Fig. 8. Channel activity and apoplastic pH of Zea mays roots.
0.1 mol m−3 nifedipine (Nif ), and LaCl (La3+) were added to the
3
medium and the kinetics of the apoplastic pH measured. Lines represent
the moment nifedipine or LaCl reaches the root (see Materials and
3
Methods). Representative curves of four experiments, each. The final
pH-changes were 0.6 to 0.9 (Nif ), and 0.13 to 0.09 (LaCl ).
3
The apoplastic spaces of a plant are manifold and their
ionic milieus are usually difficult to gain access to. In
spite of this, ion-selective microelectrodes have been
applied successfully to measure apoplastic free ion concentrations in leaves. Bowling (1987) measured the apoplastic
activities of K+ and Cl− in the leaf epidermis of
Commelina in relation to stomatal activity. Rhythmic and
light-dependent K+- and Cl−-activities in the pulvini of
Samanea and Phaseolus were measured by Lee and Satter
(1989), by Zucker et al. (1989), and by Starrach and
Mayer (1989), respectively. More recently, the ratiometric
fluorescent dye technique proved a useful alternative
way to investigate the apoplastic ionic milieu (Hoffmann
et al., 1992; Hoffmann and Kosegarten, 1995; Mühling
et al., 1995; Mühling and Sattelmacher, 1997).
Since the apoplastic space of a maize root is
rather small, a controlled insertion of a blunt electrode
is not possible. Thus the placement of sharp microelectrodes within this space requires the penetration of one
or more cells. As to the performance of such experiments,
some critical questions may be asked: how does one
recognize an apoplastic recording? Since a doublebarrelled microelectrode is used, the recognition of an
apoplastic recording is unequivocal. While the ionselective barrel measures the free ion concentration plus
voltage, the voltage barrel measures the voltage only, i.e.
it measures either inside (high negative voltages) or
outside ( low negative voltages) of cells. It cannot distinguish between a cytosolic and a vacuolar position, because
the tonoplast potential is very small (Bethmann et al.,
1995). A pH-sensitive electrode can distinguish between
Apoplastic pH 993
cytosol and vacuole, a potassium electrode cannot. Thus,
taking the information from the two barrels together, the
position of the sensitive tip is always clear.
Another question is: does the insertion of the microelectrode into the cortex change the true apoplastic milieu
through cell injury? There is no doubt that the insertion
of the microelectrode into the apoplast has an invasive
component, however, not more than during any membrane potential measurement in such a cortex (cell ). As
described in Fig. 1, the microelectrode is tested first
through intracellular recordings which give information
as to the state and performance of the electrode inside a
cell. It is an accepted fact that the plasma membrane of
an impaled cell closes the leak around the electrode,
otherwise no electrical recordings would be possible.
There is no reason to argue that an electrode leaving a
cell should not be sealed off in the same manner as an
electrode entering it, provided that the impalements are
carried out with care. Injuries of the tissue do occur of
course, but they are detected right away either by a noisy
signal and/or by the response of the electrode to changes
in the test medium. Comparing the small and reproducible
responses of the apoplast with the much larger changes
at the root surface to changes in external conditions
shown here, this view is supported.
The unstirred layer, an important physiological buffer zone
In vivo, a fine film of liquid covers the root surface. The
pH and the ion concentration within that film are relevant
for uptake and for transport processes into the root
apoplast as well as into the symplast. At least in the
primary root, where no thick cuticles or suberin incrustations prevent free diffusion of ions and water, this film is
directly connected with the apoplast through micropores
(3–8 nm) and as such represents a buffering layer mediating between apoplast and the rhizosphere (Carpita et al.,
1979; Kochian and Lucas, 1983; Grignon and Sentenac,
1991). In vitro, the extent to which this (unstirred) layer
reaches into the bulk medium, depends on the buffer
capacity of the medium, and on the velocity of the local
perfusion. As Fig. 3a shows, in vitro the unstirred layer
will reach several micrometres into the bulk of the medium
when the medium is perfused at a given velocity. The
expansion of the unstirred layer will rapidly increase when
the perfusion is stopped, i.e. the entire chamber is slowly
acidified. It can be concluded, therefore, that in vivo the
pH of the usually thin water film on the root and in the
outer parts of the root cortex apoplast do not differ
much, and that changes in pH, measured on the root
surface, closely relate to those within the apoplast, at
least to those in the outer cortex layers. So, although it
may not be possible to set a certain pH for the apoplast
of Zea mays root cortex in general, because of the pHzoning (Fig. 2a), a pH of 5.1–5.6 (depending on the ionic
composition of the adhering water film) seems most likely
in the part of the root cortex investigated under the
conditions of Fig. 3b.
The question may be asked, to what extent perfusion
is a relevant condition for a root? There are two aspects.
First, in order to get information on the regulation of the
apoplastic ionic milieu, the system has to be disturbed,
regardless of its natural relevance. Secondly, there are, in
fact, conditions where the water film adhering to the root
is washed away, for example, through torrential rain or
during flooding. Also, there are plants which constantly
have at least parts of their roots in water. Thus, the
information to what extent the apoplast might respond
to such conditions, is of great interest.
The apoplast, an ion exchanger
Cell walls contain high concentrations of uronic acids
with pK values similar to that of polygalacturonic acid
(Morvan et al., 1979; Keller et al., 1980). Thus, cations
tend to be accumulated and are reversibly retained in the
cell walls, either as free hydrated ions, or they become
immobilized by various reversible mechanisms, but
remain easily exchangeable. Although the simplest way
to describe this is the ‘Donnan model’ (Pitman et al.,
1974; Ritcher and Dainty, 1989), it appears too restrictive
with respect to the exclusion of anions, because some
cations may be tightly associated with indiffusible anions,
either chemically, electrostatically or structurally, causing
charge masking. It has been suggested that the cations
thus bound could be mobilized by acidification (Grignon
and Sentenac, 1991). The opposite effect, namely the
displacement of H+ through cations, is shown in Figs 5
and 6: when the external/apoplastic concentrations of K+
or Ca2+ are increased, the apoplast becomes more acidic.
It was surprising to find that, with respect to the ability
to replace protons, Ca2+ was apparently less effective
than K+. Clearly, one factor which favours such behaviour is the presence of K+. Since in the presence of high
[ K+] many of the exchangeable protons are probably
already removed from their binding sites, addition of
Ca2+ will not result easily in further acidification. In fact,
in the presence of high external [ K+], which also affects
the [ K+] of the apoplast (Fig. 7), addition of Ca2+ may
even cause an increase in pH (Fig. 6). Whereas the high
[ K+] will reduce the Ca2+-effect, the latter observation
may be attributed to the addition of chloride ions together
with the Ca2+: when Cl− is exchanged for the nonpermeable gluconate, Ca2+ in fact acidifies ( Fig. 6b). The
increase in pH can be explained by the activation of a
nH+/Cl−-symport, which acidifies the cytosol and
increases the apoplastic pH ( Felle, 1994). Since gluconate
is not cotransported, it has no influence on the apoplastic
pH, thus the Ca2+ could evoke its full effect. Yet another
reason for the apparent lower ability of Ca2+ to displace
994
Felle
protons may be that Ca2+, due to its two charges, will not
intrude into the apoplast as easily as K+. This latter notion
is suported by the observation that the exchange of H+
for Ca2+ is much more pronounced in the cell walls of
single cells, for instance in Chara (Ryan et al., 1992).
‘Active’ regulation of the apoplast pH
An apoplastic pH well controlled within narrow limits,
which on the one hand is acidic enough to provide enough
free protons for the uptake of anions (e.g. nitrate), but
on the other hand is not too acidic to cause thermodynamic problems (pH-gradient) for the primary pump,
appears essential for roots. In the maize root cortex these
controlled margins appear to be between pH 5 and 6.
Apart from the basically passive ion exchanger properties of the cell walls demonstrated above, the root can
control the pH of the apoplast through its plasma membrane transporters. In this context one may consider first
the H+ ATPase or proton cotransporters which will
directly contribute to the maintainance and regulation of
the apoplastic pH. In fact, when the proton pump is
inhibited or stimulated the apoplastic pH indeed increases
( Fig. 4). It was interesting, however, to observe that
neither stimulation (FC ) nor inhibition (cyanide) had the
expected massive effect on apoplastic pH, which stimulates the search for other factors. Apart from the fact
that there are other proton transporters which may influence the apoplastic pH, the regulation of cation channel
activity appears to be a rather effective way to fineregulate the apoplastic pH. In fact, it has been shown in
this work that cations can be very effective in changing
the apoplastic pH ( Figs 5, 6). What has been observed
when cations enter the apoplastic space from outside
should also hold when ions are transported into and out
of cells, a process which provides a potentially effective
way to regulate the apoplastic pH. Thus, activation of a
channel which rapidly releases ions from the cells may
well cause a transient shift in pH which, in turn, could
trigger other downstream processes. That this may indeed
be the case is shown by means of channel inhibitors. As
demonstrated in Fig. 8, the pH of the apoplast changes
right away, when cation channels are inhibited with
nifedipine and La3+, respectively. Why does the apoplastic pH increase when these channels are blocked? The
proton pump can only acidify the apoplast, when nonproton counterions compensate the transmembrane
charge transfer. When this process is disturbed, as is the
case when the channels are blocked, then this is equivalent
to a deactivation of the proton pump, leading to an
increase in apoplastic pH as demonstrated in Fig. 4b.
Acknowledgement
The financial support given by the DFG, project 717, ‘The
apoplast: compartment for storage, transport and reactions’, is
gratefully acknowledged.
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