Download Characterization of Citrate Transport through the Plasma Membrane

Plant Cell Physiol. 44(2): 156–162 (2003)
JSPP © 2003
Characterization of Citrate Transport through the Plasma Membrane in a
Carrot Mutant Cell Line with Enhanced Citrate Excretion
Takashi Ohno, Hiroyuki Koyama 1 and Tetsuo Hara
Laboratory of Plant Cell Technology, Department of Biotechnology, Faculty of Agriculture, Gifu University, 1-1, Yanagido, Gifu, 501-1193 Japan
;
overexpression of the citrate synthase gene from bacteria and
plants, increased citrate excretion from the roots. On the other
hand, overexpression of a malate dehydrogenase in alfalfa
(Tesfaye et al. 2001) causes enhanced citrate excretion from the
roots, and in turn improves growth in either the presence of Al
in the growing medium or with an Al-phosphate as the sole
phosphate source. These results indicate that the modification
of organic acid metabolisms is a target for improving organic
acid excretion from the roots of crop plants. Additionally, several studies have suggested that organic acid transport through
plasma membranes is another important factor for attaining
high organic acid excretion abilities from roots (Ryan et al.
1995).
There are two distinct patterns of organic acid excretion in
response to Al by means of differences of induction periods
between exposure to Al and the release of organic acids from
the roots. Several plants show an induction period before
releasing organic acids, whereas others release organic acids
quickly after exposure to Al (Ma 2000). The former are regulated by organic acid metabolism and the later by the membrane transport levels. Aluminum-stimulated malate efflux of
wheat is a typical example of such a quick organic acid
excretion response (Ryan et al. 1995). This efflux has been
characterized as anion-channel dependent; using anion channel
antagonists. Patch clamp analysis of plasma membrane,
isolated from the root tip cells of an Al-tolerant line, indicated
that in the short-term a kind of anion channel was stimulated by
Al (Ryan et al. 1997). Similar Al-stimulation of the anion
channel has been reported with maize (Kollmeier et al. 2001).
Moreover, anion channels are also involved in the proteoid
roots of lupin, but Al-stimulation was not required for the
excretion of citrate (Neumann et al. 1999). These results indicate that anion channels play important roles for high rates of
organic acid excretions.
On the other hand, several reports have indicated that cations were simultaneously released during the excretion of
organic acids from roots. For example, wheat and lupin
released K+ (Ryan et al. 1995) and H+ (Neumann et al. 1999,
Neumann and Römheld 1999) during the excretion of malate
and citrate, respectively. Cation transport might be necessary
for maintaining an electro-potential difference between the
plasma membrane during the release of organic acids (anion).
In order to fully understand the mechanisms of organic acid
transport through the plasma membrane, it would be important
The superior ability of citrate excretion in a carrot
(Daucus carota L.) mutant cell line, namely IPG (insoluble
phosphate grower) [Takita et al. (1999a) Plant Cell Physiol.
40: 489] cells has been characterized in terms of citrate
transport at the plasma membrane. IPG cells released
about a 20-fold increase in citrate in comparison with
malate, while the concentration of malate was only 35%
lower than that of citrate in the cell sap. Citrate excretion
was sensitive to anion channel blockers, such as niflumic
acid and anthracene-9-carboxylic acid. These results indicate that IPG cells release citrate through the plasma membrane using citrate specific anion channels. The rate of
citrate release from IPG cells was not affected by the
concentration of aluminum (0 and 50 mM), soluble Pi (0 or
2 mM) and the pH (4.5–5.6) of the medium, suggesting that
anion channels would not be regulated by such external
conditions. Citrate excretion correlated with the H+ efflux,
possibly from the action of H+-ATPase on the plasma membrane. The activity of plasma membrane H+-ATPase was
about three times higher in IPG cells than in wild-type cells,
and might be involved in the high citrate excretion ability.
Keywords: Aluminum phosphate — Anion channel — Citrate
excretion — Daucus carota — H+-ATPase.
Abbreviations: A-9-C, anthracene-9-carboxylic acid; DIDS, 4,4¢diisothiocyanostilbene-2,2¢-disulfonic acid; EA, ethacrynic acid; IPG,
insoluble phosphate grower; NA, niflumic acid; WT, wild-type.
Introduction
Organic acid excretion from roots, which plays various
roles in efficient nutrient uptake (e.g. phosphate, Schactman et
al. 1998; iron, Hirsch and Sussman 1999) and the alleviation of
metal toxicities (e.g. aluminum, Miyasaka et al. 1991, Ma
2000), is thought to be one of the most important plant traits for
adaptation to problem soils. For example, certain plant species
showing an aluminum tolerance or a high Pi-acquisition ability, such as Cassia tora L. (Ma et al. 1997) and lupin (Gardner
et al. 1981), release large amounts of organic acids from their
roots. Genetic manipulation may be one approach to introduce
such traits into crop plants. In fact, transgenic tobacco (de la
Fuente et al. 1997) and Arabidopsis (Koyama et al. 2000), with
1
Corresponding author: E-mail, [email protected]; Fax, +81-58-293-2911.
156
Citrate efflux of carrot mutant cell line
157
Table 1 Release and content of citrate and malate in IPG cells
and WT cells
Organic acids Amounts of citrate and malate (mmol g–1 FW)
Content
Released
IPG
Citrate
Malate
Fig. 1 HPLC analysis of organic acids released from IPG cells. IPG
cells were grown for 7 d with Al-phosphate (2 mM) as a sole phosphate source. The major organic acids are indicated.
to fully investigate such a relationship between organic acid
excretion and cation transport.
As we reported previously, we have been investigating
mechanisms of citrate excretion in a mutant carrot cell line
[insoluble phosphate grower (IPG)], which grows normally in
an Al-phosphate medium by enhanced citrate excretion
(Koyama et al. 1988). The IPG cell line showed a typical
altered organic acid metabolism allowing the production of
more citrate than the wild-type cells (Takita et al. 1999a). On
the other hand, this cell line has also shown a very quick
response to citrate excretion after inoculation into an Al-phosphate medium (Koyama et al. 1990). The excretion continued
linearly over a 12-h period, as with malate excretion from Altolerant wheat (Delhaize et al. 1993). In addition, the rate of
citrate excretion from IPG cells was comparable to that from
Al-tolerant maize (Pellet et al. 1995), known as one of the
highest values derived from crop plants. Both Al-tolerant lines
in wheat and maize are known to contain an altered organic
acid transport capacity in the plasma membrane. Thus, we
speculated that IPG cells could involve a similar alteration in
the plasma membrane. As noted above, the IPG cell line is
thought to be a mutant, and thus it would be a useful tool to
clarify the nature of the mechanisms of organic transport across
plasma membranes.
In the present study we have characterized the physiological mechanisms associated with citrate transport on the plasma
membrane of IPG cells. The IPG cells involve anion channels,
which relate to citrate transport, but which are not regulated by
external factors. This citrate excretion was thought to be concomitant with H+ efflux from cells.
Results
Specific citrate excretion from the IPG cells
Plants release various organic acids, such as malate, citrate, succinate and oxalate from the roots in response to Al toxicity and P deficiency (e.g. Lipton et al. 1987). To examine
whether citrate excretion from IPG cells was specific or not, we
analyzed other organic acids in the medium. After 10 d of cul-
WT
6.41±0.37 4.67±0.57
4.08±0.10 8.49±0.80
IPG
WT
0.86±0.03 0.14±0.01
0.04±0.05 0.03±0.04
Cells were pre-cultured for 5 d in a medium containing 0.5 mM of Naphosphate, and then the citrate and malate content were determined.
The pre-cultured cells were incubated in Al-phosphate medium for
10 h, and then the citrate and malate in the medium were determined.
Means ± SD (n = 3) are shown.
ture in an Al-phosphate medium, one large peak and two small
peaks were detected by HPLC analysis using a UV-absorption
detector. The large peak was characterized as citrate, and the
small peaks were identified as fumarate and malate, respectively (Fig. 1). Since the UV-absorption coefficient of fumarate was much higher than those of the others, malate was
thought to be the second major organic acid that was exuded by
the IPG cells during the long-term experiment. Thus, we measured concentrations of citrate and malate in the medium and in
the cells during a short-term experiment. During 10 h of incubation in the Al-phosphate medium, the IPG cells released
0.86 mmol of citrate per gram of fresh weight, which corresponded to about a 20-fold increase in the amount of malate
released. On the other hand, the content of malate was only
35% lower than for the citrate in the IPG cells (Table 1). Thus,
it was suggested that the IPG cells would involve a specific
transporting system of citrate at the plasma membrane.
Effect of ion environments on the citrate excretion rate of IPG cells
We analyzed citrate excretion rates when IPG cells were
incubated in various conditions to examine whether ionic environments could affect the rate of citrate excretion of IPG cells.
A linear citrate excretion was observed with IPG cells for at
least 10 h after exposure to an Al-phosphate medium, whereas
wild-type (WT) cells released very little citrate (Fig. 2, Table 1).
Thus, we compared the citrate excretion rates of IPG cells after
incubation in various conditions. The addition of soluble phosphate (2 mM) to the Al-phosphate medium caused no significant effect on citrate excretion from IPG cells at 5 h (Fig. 3).
Thus, a Pi-deprived medium would not be necessary for maintaining the rate of citrate excretion in IPG cells. To evaluate the
effects of Al ions, we determined the rate of citrate excretion
using a simple culture medium containing only CaCl2 (3 mM)
and sucrose, with the addition of AlCl3 at pH 4.5. The addition
of 50 mM of AlCl3 caused no significant change in the citrate
excretion in the IPG cells (Fig. 3), indicating that the addition
of the aluminum ion was not necessary for the activation of
citrate excretion from IPG cells. On the other hand, the pH of
the medium did have a slight affect on citrate excretion from
158
Citrate efflux of carrot mutant cell line
Fig. 2 Time course of citrate excretion from IPG (filled circle) and
wild-type (open square) cells. Both cell lines were incubated in a
medium containing colloidal Al-phosphate (2 mM Pi; 4 mM Al) at
pH 5.6 in the presence of 30 mM MES buffer. Means and SD are
shown (n = 3).
Fig. 3 Effects of soluble Pi and soluble Al on citrate excretion of IPG
cells. IPG cells were incubated for 5 h in an Al-phosphate medium
(see Fig. 2 caption) in the presence (filled column) or absence (open
column) of 2 mM of soluble phosphate (left), or incubated in 3 mM
CaCl2 solution [pH 4.5, 3% (w/v) sucrose] in the presence (filled column) or the absence (open column) of 50 mM of AlCl3 (right). Means
of three replications and SD are illustrated.
the IPG cells. Citrate excretion at pH 4.5 was significantly
lower than that at pH 5.6. The rate of citrate excretion at pH 4.5
maintained levels of about 80% and 95% relative to those at
pH 5.6 and pH 5.0, respectively (Fig. 4).
Effects of anion channel blockers on citrate excretion
Citrate exists in the cytoplasm as an anion and therefore
excretion could be mediated with anion-permeable channels.
To investigate this possibility, we examined the effects of four
anion channel blockers; niflumic acid (NA), anthracene-9carboxylic acid (A-9-C), ethacrynic acid (EA) and 4,4¢diisothiocyanostilbene-2, 2¢-disulfonic acid (DIDS), on citrate
excretion from IPG cells. To minimize secondary effects (e.g.
toxicity) of the anion channel blockers, we added each
chemical at concentrations of 50 mM, which were similar to
those used in other studies (Ryan et al. 1995, Kawachi et al.
2002). Among the four blockers, NA and A-9-C showed strong
inhibitory effects on citrate excretion (60–70%; Fig. 5). On the
Fig. 4 Effects of medium pH on citrate excretion of IPG cells. IPG
cells were incubated for 5 h in 3 mM of CaCl2 solution at different pH
values (4.5, 5.0 and 5.6). Means and SD are shown (n = 3).
Fig. 5 Effects of anion channel blockers on citrate excretion of IPG
cells. IPG cells were incubated for 10 h in an Al-phosphate medium
(see Fig. 2 caption) in the presence or absence of an anion channel
blocker (50 mM). NA, niflumic acid; A-9-C, anthracene-9-carboxylic
acid; EA, ethacrynic acid; and DIDS, 4,4¢-diisothiocyanostilbene-2,2¢disulfonic acid. Means and SD of three replications are shown.
other hand, EA and DIDS inhibited citrate excretion at only
40% and 10%, respectively. This trend was very similar to the
effect of these inhibitors on the Al-stimulated malate efflux of
wheat (Ryan et al. 1995), known as a typical example of the
anion channel mediated organic acid efflux. Thus, we could
infer that anion channels were involved in the citrate efflux
mechanism of IPG cells.
Cation efflux during citrate excretion
By maintaining electronic neutrality across the plasma
membrane, the loss of citrate anions from the cytoplasm would
be balanced by an equivalent uptake of anions or a loss of cations. To determine this factor’s effect on citrate excretion from
IPG cells, we analyzed the efflux of K+ and H+, which are
known to associate with organic acid excretion in some plant
species, in the medium. As shown Fig. 6A, IPG cells released
citrate linearly for at least 3 h after being inoculated into a simple culture medium, whereas WT cells released very small
amounts. The concentration of K+ in the medium reached about
400 mM just after inoculation in both cell types, and then
Citrate efflux of carrot mutant cell line
159
Fig. 7 Effect of vanadate on citrate excretion of IPG cells. IPG cells
were incubated in an Al-phosphate medium (see Fig. 2 caption) for
10 h in the presence of various concentrations of vanadate. Means and
SD of three replications are shown.
Fig. 6 Changes in H+ and K+ concentrations in the medium during
the citrate excretion of IPG (closed symbols) and wild-type (open symbols) cells. IPG and wild-type cells were incubated in 3 mM of CaCl2
solution [pH 5.6, 3% (w/v) sucrose] for a total of 3 h. Released citrate
(circles) and H+ (triangles) (panel A), or K+ (squares in panel B) were
quantified at the appropriate time. Means and SD of three replications
are shown.
sharply decreased in the IPG cell medium as a function of time
(Fig. 6B). Thus, the efflux of K+ was not associated with the
citrate efflux in terms of maintaining electronic chemical balance. On the other hand, the H+ concentration in the IPG cell
medium increased for at least 3 h after inoculation. Under a
neutral pH, such as in cytosol, almost all of the citrate is
thought to exist as trivalent anions. On the other hand, pH of
the simple medium ranged from 4.5 to 5.6 during the first 3 h,
and thus the citrate would be changed into divalent anions by
incorporating H+ ions after the excretion. Under these conditions, if the citrate release was accompanied by an equivalent
H+ efflux (i.e. mol ´3), a portion of the H+ (i.e. mol ´1) would
be absorbed by the citrate ion, and thus a 1 : 2 relationship
could be expected between the citrate efflux and the H+ efflux.
As expected, the ratio of increase in H+ to that of citrate was
nearly 2 to 1 (Fig. 6A). Further, the application of vanadate, an
inhibitor of plasma membrane H+-ATPase, inhibited citrate
excretion (Fig. 7). From these results, we inferred that the citrate efflux from IPG cells was associated with H+ efflux.
H+-ATPase activities in the plasma membrane
To further characterize the mechanism of citrate efflux of
IPG cells, we compared plasma membrane H+-ATPase activities between IPG and WT cells. First, we compared a series of
marker enzyme activities in the microsomes. The activities of
Cyt-c oxidase, a marker enzyme of the mitochondria inner
membrane, and NADH Cyt-c reductase, a marker enzyme of
the endoplasmic reticulum membrane, were similar between
the IPG and the WT cells. On the other hand, the activity of
nitrate-sensitive ATPase, a marker enzyme of the tonoplast,
was also similar between the IPG and the WT cells. Under
these conditions, the vanadate-sensitive ATPase activities in the
IPG and the WT cells were 0.31 and 0.25 mmol min–1 mg
protein–1, respectively (Table 2). Vanadate-sensitive ATPase is
known as a marker enzyme of plasma membrane. There were
at least two possible explanations for the higher activities
found in the microsomes of the IPG cells. One is that IPG cells
contain higher activities in this enzyme in the plasma membrane, and the other is that the relative amounts of plasma
membrane in the microsomes could be higher in the IPG than
in the WT cells. To investigate these possible explanations, we
purified plasma membranes from each cell type and then measured the activities of the enzymes. We used an aqueous polymer two-phase system, which is known to be one of the most
reliable methods for isolating highly purified plasma membrane. As shown in Table 2, the activities of the vanadate-sensitive ATPase were at least two times more concentrated in the
plasma membrane fractions, but the activities of the other
organelle membranes decreased below 10%. Under these
experimental conditions, we found that the vanadate-sensitive
ATPase activity in the plasma membrane was about three times
higher in IPG cells than in WT cells. Thus, we inferred that
IPG cells contained higher levels of H+-ATPase activities in the
plasma membrane.
160
Citrate efflux of carrot mutant cell line
Table 2 Specific activities of membrane-binding proteins in microsomes and plasma membrane
vesicles isolated from IPG and wild-type (WT) cells
Enzymes
Activities of enzymes (mmol min–1 mg protein–1)
Microsomes
Plasma membrane vesicles
IPG
WT
IPG
WT
Vanadate-sensitive ATPase
Nitrate-sensitive ATPase
0.31±0.00
0.23±0.02
0.25±0.01
0.21±0.02
1.50±0.09
0.00±0.00
0.50±0.05
0.02±0.01
NADH Cyt-c reductase
0.81±0.00
1.01±0.15
0.09±0.09
0.00±0.00
Cyt-c oxidase
0.30±0.03
0.23±0.01
0.00±0.00
0.00±0.00
Means ± SD (n = 3) are shown.
Discussion
The rate of organic acid excretion from plant roots is controlled by organic acid productivity and the transport level at
the plasma membrane (Neumann et al. 1999). As we reported
previously, IPG cells continuously release citrate into an Alphosphate medium for at least 10 d after inoculation (Koyama
et al. 1990). Such a long-term release would relate to an altered
organic acid metabolism, a combination of higher mitochondrial citrate synthase activity and lower cytosolic NADPspecific isocitrate dehydrogenase activity compared with WT
cells, and in turn to producing more citrate than WT cells
(Takita et al. 1999a). In the present study, we have characterized the mechanism of citrate transport through the plasma
membrane in IPG cells, which related to a high rate of citrate
excretion within the short term (Fig. 2). Citrate excretion was
sensitive to the addition of anion channel blockers (Fig. 5). The
inhibitory effects of NA and A-9-C were strong, but EA
showed a moderate inhibitory effect. On the other hand, DIDS
showed no effects. Since A-9-C and DIDS are often used as
potent S-type and R-type anion channel blockers, it can be
inferred that S-type anion channels are mainly involved in
citrate excretion in IPG cells. The trend of inhibitory effects of
anion channel blockers was very similar to the inhibitory
effects on the malate efflux of aluminum-tolerant wheat (Ryan
et al. 1995) and the citrate efflux of lupin (Neumann et al.
1999), despite NA having no effect on lupin. Thus, similar
anion channels would be involved in the high abilities for
organic acid efflux from plants, but either the specificity for the
organic acid or the activating mechanisms could be different.
When exposed to Al, malate, citrate and oxalate were
released from Al-tolerant lines of wheat (Ryan et al. 1995),
maize (Pellet et al. 1995) and an Al-tolerant species of buckwheat (Zheng et al. 1998). These results indicate that anion
channels have specificity to organic acids. In this study, we
found that IPG cells excreted only citrate, and that the internal
concentration of citrate was similar to that of malate (Fig. 1,
Table 1), suggesting that the anion channels of IPG cells show
specific permeability to citrate. On the other hand, the anion
channels of IPG cells would be different from those in Al-
tolerant lines or species in terms of Al-stimulation. Addition of
Al to the external medium is necessary for organic acid release
from Al-tolerant lines and species, whereas IPG cells excreted
citrate into the medium without the addition of Al (Fig. 3, 4,
6A). Similar Al-free excretion has been reported with citrate
release from the cluster root of white lupin (Neumann et al.
1999) and Pi-deprived alfalfa (Lipton et al. 1987). Although the
mechanism involved in Al-stimulation has not been fully elucidated, a candidate for cDNA encoding of a malate permeable
channel was isolated from an Al-tolerant line of wheat using
a cDNA subtraction technique (Sasaki et al. 2002). A gene
manipulation study using oocytes suggested that an anion channel encoded by cDNA showed high selectivity to malate in
comparison with citrate, and could contain an Al-responsible
domain in itself. Thus, anion channels of IPG cells may lack
the domain required for Al-stimulation. However, further analyses are needed to determine the structure of the anion channel
of IPG cells.
Organic anion transport through the plasma membrane
often coincides with cation transport and is thought to play a
role in maintaining charge balance for the release of organic
anions. For example, the Al-stimulated organic acid release
(Ryan et al. 1995) and the mugineic acid release (Sakaguchi et
al. 1999) from barley were concomitant with K+ efflux. On the
other hand, H+ exclusion is associated with an organic acid
release from cluster roots of white lupin, in response to Pideficiency (Johnson et al. 1996, Neumann et al. 1999,
Neumann and Römheld 1999). In the present study, we found
that citrate release in IPG cells was concomitant with H+ efflux,
but not potassium (Fig. 6). Our results cannot allow for the
conclusion that H+ efflux is balanced electro-chemically with
citrate efflux through the plasma membrane because the IPG
cells showed a high rate of K+ uptake (Fig. 6). Further investigations, such as determination of other ion transports during
the citrate excretion, will be needed to clarify whether citrate
transport balances H+ efflux or not.
H+ exclusion through the plasma membrane is mostly
dependent on plasma membrane H+-ATPases. As shown in
Table 2, the activity of this enzyme, which corresponds to
vanadate-sensitive activity, showed a three-fold increase in IPG
Citrate efflux of carrot mutant cell line
cells relative to WT cells. In addition, in vivo citrate excretion
was sensitive to vanadate, and about 50% of excretion was
inhibited by 0.5–1 mM of vanadate (Fig. 7). Very recently, a
similar result was reported with a proteoid root of white lupin
(Yan et al. 2002). Vanadate-sensitive ATPase activity was
found to be 2–2.5-fold higher in Pi-deprived proteoid root than
in the others, and addition of 1 mM of vanadate inhibited acidification by the proteoid root. Thus, it can be inferred that an
increased activity of this enzyme is necessary for enhanced citrate excretion, at least in some plant species.
As we have reported, IPG cells have improved citrate productivity in which at least two enzymes are altered at the transcript level (Takita et al. 1999b, Kihara et al. 2003). The
present study indicates that both the citrate permeable anion
channel(s) (Table 1) and the plasma membrane H+-ATPase(s)
(Table 2) are altered in IPG cells, but the molecular mechanisms remain to be clarified. This combined pattern is very
similar to the possible mechanism of enhanced citrate excretion from the proteoid root of lupin (Neumann et al. 1999).
Very interestingly, IPG cells have shown another characteristic, namely a superior phosphate uptake (Koyama et al. 1992),
which is also similar to the proteoid root of lupin. It would be
interesting to identify the molecular mechanism by which IPG
cells have acquired such a series of characteristics, closely
related to the characteristics in the proteoid root of lupin. Further investigations are clearly needed to clarify this factor.
Materials and Methods
Plant materials and culture conditions
The mutant cell line (IPG) of carrot and the wild-type cell line
(WT) used in the present study were identical to those used in our previous reports (e.g. Koyama et al. 1988). Both cell types have been
maintained as suspension cultures (Takita et al. 1999a) in liquid R2
medium (Ohira et al. 1973). To investigate organic acid release into
medium during the growth period, IPG cells were grown for 7 d in R2
medium containing 2 mM of Na-phosphate or Al-phosphate as the sole
phosphate source, as described previously (Koyama et al. 1990). On
the other hand, cells were precultured in R2 medium containing
0.5 mM Na-phosphate for 5 d.
Determination of the rate of citrate excretion
Each 4 g of cells was inoculated into 100 ml of various kinds of
exudate collection media, and then shaken on a rotary shaker
(120 rpm) at 25°C. An Al-phosphate medium containing colloidal Alphosphate was prepared by adding 4 mM of polymeric AlCl3 (stock
solution; 100 mM, adjusted to pH 4.0 by adding NaOH) to the R2
medium in the presence of 30 mM of MES-buffer (pH 5.6). After autoclaving, soluble Pi (Pi < 0.01 mM) was unable to be determined in this
medium (Koyama et al. 1988). A simple medium was prepared by
adding 3% (w/v) sucrose to 3 mM CaCl2 solution, and then adjusting
to the appropriate pH. This medium was known to enhance Al-toxicity
in cultured cells (Ikegawa et al. 2000). The effect of soluble Pi was
determined using the Al-phosphate medium by adding 2 mM of Naphosphate after autoclaving. The effects of pH (pH 4.5, 5.0 and 5.6)
and soluble Al (pH 4.5, 50 mM AlCl3 from 1 mM stock solution) were
determined using a simple medium. Anion channel antagonists, NA,
A-9-C, EA and DIDS were added to the Al-phosphate medium to give
161
a final concentration of 50 mM. Scarcely soluble inhibitors were first
dissolved in a small amount of ethanol and then diluted in the Alphosphate medium. The effect of vanadate was determined in the Alphosphate medium containing various concentrations of vanadate.
Citrate and malate in the exudates collection medium or the cell
extracts were measured separately by an enzymatic cycling method
according to Hampp et al. (1984), with minor modifications, as
described previously (Takita et al. 1999a). HPLC analysis was carried
out using a specific column for organic acid analysis (Shodex KC-811,
Showa Denko, Tokyo, Japan) and a UV detector (Uvidec-100, JASCO,
Tokyo, Japan), as described previously (Koyama et al. 1988).
H+ and K+ efflux
Each 4 g cell was inoculated into the simple medium (3 mM
CaCl2, 3% sucrose) at an initial pH of 5.6. The culture medium was
prepared as described above. The pH of the culture medium was monitored using a pH meter, and then the H+ concentration was calculated
using the following equation: [H+] (mol l–1) = 10–pH. The concentration of K+ was determined using an emission spectrophotometer (170–
40; Hitachi, Tokyo, Japan).
Isolation of plasma membrane
Plasma membranes were purified from microsomes with an
aqueous polymer two-phase system, as described by Yoshida et al.
(1983) and Yoshida and Uemura (1986), with minor modifications. All
manipulations were conducted on ice with prechilled buffers. Cells
(30 g) were ground in 90 ml of a 25 mM potassium phosphate buffer
(pH 7.8) containing 0.25 M mannitol, 1.5% (w/v) polyvinylpyrrolidone, 0.1% (w/v) BSA (fatty acid free, Sigma), 1 mM dithiothreitol,
2 mM EGTA and 2 mM EDTA-2Na, using a mortar and pestle. Cell
debris was eliminated by passing through four layers of gauze, followed by centrifugation at 13,000´g (0°C) for 10 min. The microsomes were derived from the supernatant by centrifugation at
170,000´g (0°C) for 35 min, and then washed by the same centrifugation after being suspended in a 10 mM potassium phosphate buffer
(pH 7.8) containing 0.25 M sorbitol and 1 mM DTT. Microsomes were
resuspended in 2 ml of the same buffer, which was used for washing,
and then added to an aqueous polymer two-phase system consisting of
6.4% (w/v) polyethyleneglycol 3350, 6.4% (w/v) Dextran T500,
0.25 M sorbitol, 1 mM DTT and 30 mM NaCl in a 10 mM potassium
phosphate buffer (pH 7.8). The above solution was shaken gently,
placed at 0°C for 30 min and then centrifuged at 750´g (0°C) for
4 min. The upper phase, which contained plasma membrane vesicles,
was mixed with the lower phase of the above solution. This step was
repeated three times to increase purity of the plasma membrane fraction. The plasma membrane vesicles in the upper phase were collected
by centrifugation at 170,000´g (0°C) for 35 min, and washed twice
with 25 mM HEPES-BTP (pH 7.2) containing 0.25 M sorbitol. The
plasma membrane fraction was re-suspended in 1 ml of the same
buffer in the presence of 2 mM DTT and kept at –80°C. The activity
of the ATPase was measured according to the method of Uemura and
Steponkus (1989). Specific activities for the vanadate and nitratesensitive ATPases (plasma membrane and tonoplast markers) were
measured in the presence of 0.1 mM vanadate and 100 mM KNO3,
respectively. Antimycin-A-insensitive NADH Cyt-c reductase (endoplasmic reticulum membrane marker) and Cyt-c oxidase activities
(mitochondria inner membrane marker) were measured according to
Uemura and Yoshida (1983). Protein was assayed by the method of
Bradford (1976) using BSA as the standard.
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162
Citrate efflux of carrot mutant cell line
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(Received August 9, 2002; Accepted November 26, 2002)