Journal of Experimental Botany, Vol. 49, No. 318, pp. 13–19, January 1998
Control of phosphate transport across the plasma
membrane of Chara corallina
Tetsuro Mimura1, Robert J. Reid2,3 and F. Andrew Smith2
1 Biological Laboratory, Hitotsubashi University, Naka 2–1, Kunitachi, Tokyo 186, Japan
2 Centre for Plant Membrane Biology and Department of Botany, University of Adelaide, Adelaide 5005,
Australia
Received 12 May 1997; Accepted 20 August 1997
Abstract
This paper examines the control of phosphate uptake
into Chara corallina. Influxes of inorganic phosphate
(Pi) into isolated single internodal cells were measured
with 32Pi. Pretreatment of cells without Pi for up to
10 d increased Pi influx. However, during this starvation the concentrations of Pi in both the cytoplasm
and the vacuole remained quite constant. When cells
were pre-treated with 0.1 mM Pi, the subsequent influx
of Pi was low. Under these conditions the Pi concentration in the cytoplasm was almost the same as that of
Pi-starved cells, but vacuolar Pi increased with time.
Transfer of cells from medium containing 0.1 mM Pi
to Pi-free medium induced an increase of Pi influx
within 3 d irrespective of the concentration of Pi in the
vacuole.
During Pi starvation, neither the membrane potential
nor the cytoplasmic pH changed. Manipulation of the
cytoplasmic pH by weak acids or ammonium decreased
the Pi influx slightly.
Pi efflux was also measured, using cells loaded with
32Pi. Addition of a low concentration of Pi in the rinsing
medium rapidly and temporarily induced an increase
in the efflux.
The results show that Pi influx is controlled by
factors other than simple feedback from cytoplasmic
or vacuolar Pi concentrations or thermodynamic driving forces for H+-coupled Pi uptake. It is suggested
that uptake of Pi is controlled via the concentration
of Pi in the external medium, through induction or
repression of two types of plasma membrane Pi
transporters.
Key words: Chara corallina, membrane transport, phosphate influx, phosphate starvation.
Introduction
Inorganic phosphate is one of the essential nutrients for
plant growth. In the natural environment, Pi concentrations in soil are usually extremely low ( less than 10 mM )
(Marschner, 1995), and plants are often potentially under
Pi deficiency. It is well known that plant cells can cope
with Pi deficiency by changing Pi transport activities
across the membrane, i.e. by increasing V
and/or
max
decreasing K (Bieleski, 1973; Bieleski and Ferguson,
m
1983). In higher plant cells, Pi uptake is considered to be
driven by the electrochemical potential gradient for protons which is generated by the plasma membrane H+ATPase ( Ullrich-Eberius et al., 1981, 1984; Sakano et al.,
1992). Recently genes of the Pi transporter in the plasma
membrane have been isolated from various plants
(Harrison and van Buuren, 1995; Umesh et al., 1996).
They have shown that when the external Pi supply was
limited, the number of Pi transporter molecules increased
and/or different types of Pi transporter were induced in
plant tissues. However, it is not yet known how the plant
cell detects changes in Pi supply and controls expressions
of Pi transporters.
There are many reports dealing with a relationship
between Pi supply and Pi transport of the plasma membrane. Some have included measurements of the changes
in Pi concentrations of both the cytoplasm and the
vacuole (Lee and Ratcliffe, 1983; Rebeille et al., 1983;
Mimura et al., 1990). They showed that although the
vacuolar Pi concentration changed in response to Pi
3 To whom correspondence should be addressed. Fax: +61 8 8232 3297. E-mail: [email protected]
Abbreviations: DMO, dimethyloxazolidine-2,4-dione; Pi, inorganic (ortho)phosphate.
© Oxford University Press 1998
14
Mimura et al.
supply, the cytoplasmic Pi concentration remained constant. This suggests that the Pi transport activity at the
plasma membrane may be related not to the cytoplasmic
Pi concentration but the vacuolar concentration, although
precisely how signals from the vacuole might be sensed
at the plasma membrane is not known.
Until now, most work on cellular Pi transport mechanisms related to Pi supply has involved intact plants or
proliferating cultured cells as experimental material
(Mimura, 1995). In intact plants, Pi in the cells is
transported from root to shoot or from older cells to
younger cells. In dividing cultured cells, Pi is always
diluted in the daughter cells when under Pi deficiency. In
both cases, the vacuolar Pi pool must be affected not
only by the external Pi supply, but also by the stage of
cell growth and division. In the present study, this complication has been overcome through the use of isolated
mature Chara internodal which do not grow (or grow
only very slowly), divide or differentiate. Using such cells,
it has been possible to show that transport activity of Pi
across the plasma membrane is independent of the concentration of Pi in the cytoplasm or in the vacuole. Other
possible factors which might control Pi transport across
the plasma membrane have also been examined.
Materials and methods
Plant material and culture
Chara corallina was cultured in an experimental pond (outdoors,
growth solution undefined ), or indoors in plastic tanks on a
substrate of garden soil and river sand with unbuffered solution
containing 0.1 mM K SO , 1 mM NaCl and 0.5 mM CaCl
2 4
2
under fluorescent lamps with a 16/8 h light/dark cycle at room
temperature (around 22 °C ). Cells from the different cultures
were equivalent except that the cells from the outside pond
tended to have lower vacuolar Pi concentrations due to the
lower concentration of Pi in the growth solution. Individual
internodal cells were excised from shoots at least 1 d before
experiments and stored in an artificial pond water (APW )
composed of 1 mM NaCl, 0.1 mM KCl and 0.5 mM CaCl . In
2
some measurements, cells were transferred 1 d after isolation
into APW buffered with 2 mM 2-(N-morpholino)ethanesulphonic acid (MES), whose pH was adjusted to 6.0 with NaOH.
They were then stored until required for flux measurements
with daily solution changes.
Measurement of Pi content
Inorganic phosphate in internodal cells was extracted using two
methods. (1) When 32Pi flux was measured, appropriate aliquots
of vacuolar sap were collected with a micro-capillary tube after
incubation in the medium containing 32Pi. (2) For the
measurement of intracellular distribution of Pi, vacuolar sap of
internodal cells was first replaced with an artificial cell sap
composed of 100 mM KCl, 30 mM NaCl, 10 mM MgCl , and
2
2 mM CaCl by vacuolar perfusion (Tazawa et al., 1987).
2
Isolated vacuolar sap was diluted with deionized water as the
vacuolar fraction. The rest of the cell was put into water as the
cytoplasmic fraction. The cytoplasmic fraction contained not
only the cytosolic Pi but also Pi in chloroplasts and the cell
wall. Both samples were boiled for 7 min. Pi in each sample
was measured according to the method of Bencini et al. (1983).
NaH PO was used as a standard.
2 4
Measurement of Pi influx
Pi influx was measured by incubating cells in solutions
containing 32Pi (specific activity approximately 10 Bq nmol−1)
in buffered APW for 20 min. The concentration of Pi in the
incubation media was adjusted with addition of non-radioactive
NaH PO . After incubation, cells were washed twice with APW
2 4
(total 2 min), then blotted gently. A sample of vacuolar sap
was removed from each cell for the measurements of both Pi
concentration and radioactivity in the vacuole. The rest of the
cell was put into scintillation fluid and the radioactivity was
measured in a liquid scintillation counter (LS3801; Beckman,
USA).
Measurement of Pi efflux
For the measurement of Pi efflux, internodal cells were loaded
with 32P solution for between 7 and 10 d. Individual cells were
put into a syringe and repeatedly rinsed with buffered APW.
Efflux was calculated from the radioactivity in the rinse
solutions. Rates were normalized for the initial 1 min as 100%
in order to compare individual cells.
Measurement of membrane electrical potential
The membrane potential difference of internodal cells was
measured with conventional KCl-filled glass microelectrodes
inserted into the vacuole of internodal cells. The cells were
mounted in a perpex holder and the extracellular solution was
perfused using a peristaltic pump.
Measurement of cytoplasmic pH
Cytoplasmic and vacuolar pH were measured in intact cells by
distribution of 14C-DMO between the bathing solution and the
cytoplasm ( Walker and Smith, 1975).
The cytoplasmic pH of internodal cells was manipulated as
follows. Cells were treated with 1.0 mM butyric acid in APW
pH 5 for 20 min to lower the cytoplasmic pH (Reid et al.,
1989), or were treated with 0.2 mM NH Cl in APW pH 6 for
4
2 h to increase the cytoplasmic pH (Smith, 1980).
Results
Relationship between Pi concentrations of the cytoplasm
and vacuole
Higher plant cells can keep the Pi concentration of the
cytoplasm almost constant under various Pi nutrition
regimes by utilizing the vacuolar Pi as a reservoir
(Mimura, 1995). To ascertain if the same mechanism
works in internodal cells of Chara, Pi concentrations of
the cytoplasm and the vacuole were measured in cells
isolated from various cultures ( Fig. 1). The cytoplasmic
Pi concentrations were mostly between 10–15 mM, but
the vacuolar Pi concentrations were distributed in a wider
range: 1–8 mM.
Changes in Pi influx during incubation with or without Pi
It is well known that the Pi influx into higher plant cells
increases under Pi deficiency and decreases under a high
Control of phosphate influx
15
cells transferred from 0 to 0.1 mM showed a large
decrease.
Changes in Pi concentrations of the cytoplasm and vacuole
Fig. 1. Relationship between Pi concentrations in the cytoplasm and in
the vacuole of Chara cells isolated from various cultures.
Pi supply (Bieleski and Ferguson, 1983; Mimura et al.,
1990). The changes in Pi influx into isolated internodal
cells pretreated in buffered APW containing 0, 0.1 and
5 mM NaH PO were measured. Figure 2 shows the Pi
2 4
influx measured over 20 min after transfer from these
pretreatment regimes to 0.1 mM 32Pi in APW pH 6. Influx
decreased in the first 24 h for all treatments. In cells that
had been incubated without Pi, the influx then increased,
while in the cells incubated in 0.1 mM or 5 mM Pi, the
influx continued to decrease; there was no difference
between the 0.1 mM and the 5 mM treatments. After 10 d
incubation, cells in the medium without Pi were transferred into the medium containing 0.1 mM Pi and vice
versa. Within 3 d, 32Pi influx into cells moved from 0.1
to 0 mM Pi showed a large increase while Pi influx in
Fig. 2. Changes in Pi influx of isolated Chara internodal cells during
incubation with or without Pi. Open circles: cells incubated without Pi
for the first 10 d. Open squares: cells pretreated with 0.1 mM Pi for the
first 10 d. Triangles: cells pretreated with 5 mM Pi for the first 10 d.
After 10 d pretreatment, cells in the medium without Pi were transferred
into the medium containing 0.1 mM Pi and vice versa.
In order to analyse the cause of the changes in Pi uptake
activity during Pi starvation, the concentrations of Pi in
the cytoplasm and vacuole were measured first. Figure 3a
shows that when internodal cells were incubated in solutions containing 0, 0.1 or 5 mM Pi, the concentration of
Pi in the cytoplasm remained fairly constant, with significant differences in concentration only becoming apparent after 6 d pre-treatment in the different Pi solutions.
The concentration of Pi in the vacuole also remained
constant in solutions containing no Pi, which suggests
that there is no mechanism for removal of Pi from the
vacuole under these conditions. The concentration of Pi
in the vacuole did increase markedly when either 0.1 mM
or 5 mM Pi was added to the external solution ( Fig. 3b).
Cells used for this experiment were from the same
preparation as that in Fig. 2 and it is therefore possible
Fig. 3. Pi concentrations in the cytoplasm (a) and in the vacuole (b)
during incubation with or without Pi. Symbols are the same as those
in Fig. 2.
16
Mimura et al.
to compare the response of influx to different concentrations of Pi in the medium with changes in Pi in the
intracellular compartments. When Pi was present in the
external medium, vacuolar Pi increased and influx fell.
However, when Pi was absent from the medium, the
vacuolar Pi remained relatively constant and Pi influx
was strongly stimulated. The presence or absence of Pi in
the medium did not affect the cytoplasmic Pi concentration, at least for the first 3 d, and therefore the changes
in influx during this period cannot be attributed to
changes in cytoplasmic Pi.
Dependence of Pi influx on Pi concentrations in both Pi-rich
and Pi-starved cells
Measurement of the dependence of Pi influx on Pi concentration showed that multiple transport mechanisms may
be involved in Pi uptake into Chara (Fig. 4a, b). One
system had a high affinity for Pi and the other a lower
affinity. In cells pretreated in 0.1 mM Pi, the high affinity
system had K =4 mM, V =2.7 nmol m−2 s−1 and the
m
max
low affinity system had K =220 mM, V =
m
max
17 nmol m−2 s−1. When cells were pretreated without Pi
for 7 d, the K of both systems did not change significms
antly, but the V
of the higher affinity system increased
max
approximately 4-fold and that of the lower affinity system
increased approximately 2-fold.
Measurements membrane PD and cytoplasmic pH in Pi-rich
and Pi-starved cells
The experiments described above showed that Pi influx
was unrelated to the concentration of Pi in the cytoplasm.
Pi uptake in a plant cell is thought to be driven by the
electrochemical potential gradient of protons, which is
composed of the membrane potential and the pH differences between the cytoplasm and the external medium.
Accordingly, changes in the electrochemical potential
gradient during incubation with Pi might affect Pi influx.
The membrane potential of cells incubated with 0.1 mM
Pi or 0 mM Pi for 6 d were compared first and no
difference was found between the treatments (Table 1).
Cytoplasmic pH was measured under similar conditions
and again no difference was found between Pi-rich and
Pi-starved cells. Treatment of Pi-starved cells with 0.1 mM
Pi for only 2 h resulted in a small acidification of the
cytoplasm, but this may be a transient phenomenon
caused by the onset of the H+-coupled Pi transport
( Ullrich and Novacky, 1990; Sakano et al., 1992).
Uptake of Cl− into Chara is strongly influenced by
cytoplasmic pH (Sanders, 1980; Reid and Walker, 1984).
Although the DMO technique did not detect a change of
cytoplasmic pH during Pi starvation, a small change in
the cytoplasmic pH (i.e. less than 0.1) might nevertheless
affect the Pi influx. Cells were treated in solutions containing either a weak acid or a weak base under conditions
which have been previously shown to induce an increase
(NH ) or a decrease (butyrate) in cytoplasmic pH of
4
around 0.3 units (Smith, 1980; Reid et al., 1989).
Incubation with butyrate for 20 min or with NH for 2 h
4
had no significant effect on Pi influx ( Table 2). Thus,
there appears to be no relationship between the Pi influx
and cytoplasmic pH.
Table 1. Membrane potential and cytoplasmic pH of Pi-rich and
Pi-starved Chara cells
Fig. 4. (a) Dependence of Pi influx on external Pi concentrations. Open
circles: cells pretreated without Pi for 7 d. Closed circles: cells pretreated
with 0.1 mM Pi for 7 d. (b) Double reciprocal plots of (a).
Treatment
Membrane potential
(mV )a
Cytoplasmic pHb
Pi-rich
Pi-starved
Pi-starved; then
+0.1 mM Pi for 2 h
−238±4 (5)
−230±3 (7)
7.41±0.08 (9)
7.38±0.02 (10)
—
7.28±0.06 (10)
aCells were incubated with 0.1 mM Pi or without Pi for 6 d before
measurements.
bCells were incubated with 0.1 mM Pi or without Pi for 7 d before
measurements.
Control of phosphate influx
Table 2. Effect of manipulation of cytoplasmic pH on Pi influx
across the plasma membrane of Chara cells
Treatment
Alkali shift (pH 6.5)
0
Control
NH Cl (0.2 mM, 2 h)
4
Acid shift (pH 5.0)
0
Control
Butyric acid (1.0 mM, 20 min)
Pi influx (nmol m−2 s−1)
5.16±1.18 (n=5)
4.38±0.56 (n=5)a
6.27±0.30 (n=5)
5.32±0.24 (n=5)b
aCells for alkali loading were incubated for 2 d without Pi before
measurements.
bCells for acid loading were incubated for 7 d without Pi before
measurements.
Effect of external Pi on the Pi efflux
Although the Pi influx increased during Pi starvation, this
occurred in the absence of any measurable changes in the
Pi concentrations of the cytoplasm or the vacuole, the
membrane potential or the cytoplasmic pH, as shown
above. Factors that control the net uptake of Pi include
Pi efflux and the latter might be sensitive to the concentration of Pi in the external medium. Figure 5 shows a Pi
efflux curve from isolated internodal cells, normalized by
taking the efflux of the first minute as 100%. In this
experiment, cells were first washed with buffered APW and
then after 60 min the washing medium was changed to
buffered APW containing 100 mM Pi. This addition of Pi
induced a small but transient increase in efflux, the magnitude of which was similar over the range 10–500 mM Pi.
Discussion
Homeostasis of the cytoplasmic Pi concentration
In higher plant cells, the cytoplasmic Pi concentration
can be kept constant by using the vacuole as a Pi reservoir
Fig. 5. Time-dependent changes in 32Pi efflux. Pi efflux was normalized
to the efflux during washing for the initial 1 min as 100%. After 60 min,
100 mM Pi was added to the washing solution.
17
(Bieleski, 1973; Mimura et al., 1990, 1996). Figure 1
shows that Chara cells can also maintain the cytoplasmic
Pi concentration between 10 and 20 mM irrespective of
the original culture conditions. Furthermore, even when
the external Pi supply was changed, the cytoplasmic Pi
concentration remained relatively constant ( Fig. 3a). This
is in contrast to the vacuolar Pi concentration which
increased greatly with increasing Pi in the external solution ( Fig. 3b).
Changes in Pi uptake across the plasma membrane
During incubation with different concentrations of Pi, the
influx into isolated internodal cells changed significantly
( Fig. 2). Pi starvation increased the influx, and the presence of external Pi reduced the influx. It can be argued
that the decreased influx after 24 h is caused by increased
cytoplasmic Pi (all treatments). However, the continuing
increased influx in cells starved for 1–10 d cannot be
correlated with decreasing cytoplasmic Pi; thus other
factors must be involved. Since the vacuolar Pi concentration did not change significantly during Pi starvation
( Fig. 3b) it is also difficult to see how the vacuolar Pi
might control plasma membrane Pi transport activity
( Figs 2, 3). It is interesting to note that when starved
cells were supplied with Pi, the vacuolar Pi concentration
increased rapidly, but when Pi was withdrawn from P-rich
cells, the vacuolar concentration did not fall. Despite the
fact that these cells then contained high concentrations
of Pi in the vacuole, removal of Pi from the external
solution induced the same rise in influx as in Pi-starved
cells with low vacuolar Pi ( Fig. 2). Thus it has been
shown for the first time that the increase in influx caused
by starvation is independent of the concentration of Pi in
the vacuole. This does not appear to be the case following
transfer from 0 Pi to 0.1 or 5mM Pi where Pi influx was
inversely related to the vacuolar Pi concentration.
The dependence of Pi influx on external Pi concentration showed that there are two different systems in Chara
( Fig. 4); one has a high affinity for Pi and the other has
a low affinity. About 30 years ago, Smith (1968) first
reported that Pi influx into Nitella translucens cells showed
an hyperbolic saturation curve with a K for Pi of
m
approximately 100 mM. It can only be speculated that the
apparent absence of biphasic isotherms in that study is
due to constitutive differences between Chara and Nitella,
an issue which could easily be resolved experimentally.
Is Pi influx controlled by the electrochemical potential
difference for protons or by pH ?
c
The driving force for Pi influx is thought to be the
electrochemical potential gradient of protons that is set
up by the plasma membrane H+-ATPase ( UllrichEberius, 1981, 1984). Smith (1968) showed that Pi influx
into Nitella cells was inhibited by the respiratory
18
Mimura et al.
uncoupler CCCP and this might be due to an effect on
the electrochemical potential difference for protons. In
the present study, two components of the electrochemical
potential gradient of protons were measured, i.e. the
membrane potential and cytoplasmic pH (pH gradient
under constant external pH ). As shown in Table 1, there
were no significant differences in either the membrane
potential or the cytoplasmic pH during Pi starvation. The
constancy of cytoplasmic pH means that control cannot
be exerted by a change from H PO− to HPO2− vice
2 4
4
versa. Accordingly, thermodynamic control of Pi influx
through changes in proton gradients seems to be ruled
out by these experiments, at least for Chara. Nor is it
likely that changes in cytoplasmic pH exert any form of
kinetic control over Pi transport, as appears to be the
case with Cl− influx in Chara (Sanders, 1980; Smith,
1980; Reid and Walker, 1984).
While it remains uncertain about how Pi influx is
controlled, evidence has recently been found that the
starvation-induced increase in Pi influx is a Na+-coupled
transport. The detailed results of this transport system
will be shown in a forthcoming paper.
Effect of external Pi on Pi efflux
Net flux of Pi is composed of unidirectional influx and
also unidirectional efflux of Pi across the plasma membrane; other cellular membranes are also likely to be
involved. When cells of higher plants are subjected to
Pi-deficiency, the unidirectional efflux decreases (Lee
et al., 1990; Bieleski and Läuchli, 1992). When Pi was
added to the external medium, the efflux of Pi from Chara
immediately increased, but then soon returned to the
original concentration ( Fig. 5). This suggests that the
efflux mechanism can detect whether Pi is present or not
outside the cell. It is unlikely that the increased efflux was
caused by a sudden increase in cytoplasmic Pi concentrations, because 10–500 mM Pi in the external medium
showed the same effects, and in any case, increased (nonradioactive) Pi in the cytoplasm would be expected to
lower the specific activity of 32Pi and hence decrease the
efflux of 32Pi from the cell. The significant outcome of
this experiment was not so much the fact that addition
of Pi externally caused a small increase in efflux, but that
the cell is able to detect Pi in the external medium, and
this implicates external Pi as a possible control factor in
Pi transport across the plasma membrane.
Conclusions
It can be concluded that Pi influx is not controlled by
overall intracellular Pi status nor by vacuolar Pi.
Decreased influx after excision might be due to increased
cytoplasmic Pi but the long -term increases during starvation are not correlated with continuing decrease in cyto-
plasmic Pi. Since efflux of Pi appears to be low in these
cells, net uptake of Pi can also be considered to be
independent of internal Pi. On the basis of these results,
it is suggested that Pi uptake is controlled by the concentration of Pi in the external medium, possibly via induction or repression of the synthesis of plasma membrane
Pi transporters, of which there appear to be two types in
Chara. Although this may seem to be a less precise mode
of control than by intracellular demand, under natural
conditions (as opposed to the artificial manipulations
imposed here), the end result would be approximately the
same—a higher transport capacity, especially of the high
affinity system, under conditions where the Pi supply is
limiting. The slow increase in Pi influx over several days
would be consistent with the time required to synthesize
more Pi transporters. However, the possibility that Pi
influx begins to increase immediately or at least over
periods too short for the synthesis of new transporters
cannot be dismissed: at present the natural variability in
Pi influx prevents us from resolving smaller changes.
Whether this system applies to higher plants remains
to be shown, but there is already evidence consistent with
such a mechanism. Mimura et al. (1996) have shown that
barley plants grown in solutions high in Pi soon behaved
as Pi-deficient plants when the medium was changed to
one lacking Pi. Similar phenomena have been shown for
tomato (Clarkson and Scattergood, 1982) and for Azolla
(Bieleski and Läuchli, 1992).
Acknowledgements
The authors wish to thank Ms Martine Long (Adelaide
University) for her kind support during the experiments. The
visit to Australia by T.M. was supported by Grant-in-Aid for
International Scientific Research (Joint Research) by The
Japanese Ministry of Education, Science, Sports and Culture.
References
Bencini DA, Wild JR, O’Donovan GA. 1983. Linear one-step
assay for the determination of orthophosphate. Analytical
Biochemistry 132, 254–8.
Bieleski RL. 1973. Phosphate pools, phosphate transport, and
phosphate availability. Annual Review of Plant Physiology
24, 225–52.
Bieleski RL, Ferguson IB. 1983. Physiology and metabolism of
phosphate and its compounds. In: Läuchli A, Bieleski RL,
eds. Encyclopedia of plant physiology, Vol. 15A. Berlin:
Springer-Verlag, 422–49.
Bieleski RL, Läuchli A. 1992. Phosphate uptake, efflux and
deficiency in the water fern, Azolla. Plant, Cell and
Environment 15, 665–73.
Clarkson DT, Scattergood CB. 1982. Growth and phosphate
transport in barley and tomato plants during the development
of, and recovery from phosphate-stress. Journal of
Experimental Botany 33, 865–75.
Harrison MJ, van Buuren ML. 1995. A phosphate transporter
Control of phosphate influx
from the mycorrhizal fungus Glomus versiforme. Nature
378, 626–9.
Lee RB, Ratcliffe RG. 1983. Phosphorus nutrition and the
intracellular distribution of inorganic phosphate in pea root
tips: a quantitative study using 31P-NMR. Journal of
Experimental Botany 34, 1222–44.
Lee RB, Ratcliffe RG, Southon TE. 1990. 31P-NMR measurements of the cytoplasmic and vacuolar Pi content of mature
maize roots: relationships with phosphorus status and
phosphate fluxes. Journal of Experimental Botany 41, 1063–78.
Marschner H. 1995. Mineral nutrition of higher plants, 2nd edn.
London: Academic Press.
Mimura T. 1995. Homeostasis and transport of inorganic
phosphate in plants. Plant and Cell Physiology 36, 1–7.
Mimura T, Dietz K-J, Kaiser W, Schramm MJ, Kaiser G, Heber
U. 1990. Phosphate transport across biomembranes and
cytosolic phosphate homeostasis in barley leaves. Planta
180, 139–46.
Mimura T, Sakano K, Shimmen T. 1996. Studies on the
distribution, re-translocation and homeostasis of inorganic
phosphate in barley leaves. Plant, Cell and Environment
19, 311–20.
Rebeille F, Bligny R, Martin J-B, Douce R. 1983 Relationship
between the cytoplasm and vacuole phosphate pool in Acer
pseudoplatanus cells. Archives of. Biochemistry and Biophysics
225, 143–8.
Reid RJ, Smith FA, Whittington J. 1989. Control of intracellular
pH in Chara corallina during uptake of weak acid. Journal of
Experimenal Botany 40, 883–91.
Reid RJ, Walker NA. 1984 Control of Cl influx in Chara by
internal pH. Journal of Membrane Biology 78, 157–62.
19
Sakano K, Yazaki Y, Mimura T. 1992. Cytoplasmic acidification
induced by inorganic phosphate uptake in suspensioncultured Catharanthus roseus cells. Plant Physiology 99,
672–80.
Sanders D. 1980. Control of Cl influx in Chara by cytoplasmic
Cl− concentration. Journal of Membrane Biology 52, 51–60.
Smith FA. 1968. Active phosphate uptake by Nitella translucens.
Biochimica et Biophysica Acta 126, 94–9.
Smith FA. 1980. Comparison of the effects of ammonia and
methylamine on chloride transport and intracellular pH in
Chara corallina. Journal of Experimental Botany 31, 597–606.
Tazawa M, Shimmen T, Mimura T. 1987. Membrane control in
the characeae. Annual Review of Plant Physiology 38, 95–117.
Ullrich CI, Novacky AJ. 1990. Extra- and intracellular pH and
membrane potential changes induced by K+, Cl−, H PO−,
2 4
and NO− uptake and fusicoccin in root hairs of Limnobium
4
stoloniferum. Plant Physiology 94, 1561–7.
Ullrich-Eberius CI, Novacky A, Fischer E, Lüttge U. 1981,
Relationship between energy-dependent phosphate uptake
and the electrical membrane potential in Lemna gibba G1.
Plant Physiology 67, 797–801.
Ullrich-Eberius CI, Novacky A, van Bel AJE. 1984. Phosphate
uptake in Lemna gibba G1: energetics and kinetics. Planta
161, 46–52.
Umesh SM, Pardo JM, Raghothama KG. 1996. Phosphate
transporters from the higher plant Arabidopsis thaliana.
Proceedings of the National Academy of Sciences, USA 93,
10 519–23.
Walker NA, Smith FA. 1975. Intracellular pH in Chara corallina
measured by DMO distribution. Plant Science Letters
4, 125–32.