Download Cycloprodigiosin Hydrochloride Inhibits Acidification of the Plant

Plant Cell Physiol. 40(2): 143-148 (1999)
JSPP © 1999
Cycloprodigiosin Hydrochloride Inhibits Acidification of the Plant Vacuole
Tsuyoshi Nakayasu1, Keiko Kawauchi, Hajime Hirata and Teruo Shimmen
Department of Life Science, Faculty of Science, Himeji Institute of Technology , Harima Science Park City, Hyogo, 678-1297 Japan
The effect of cycloprodigiosin hydrochloride (cPrGHC1), obtained from the marine bacterium Pseudoalteromonas denitrificans, on acidification of the vacuole was
studied in Characeae cells. In internodal cells of Nitella,
the plasma membrane of which had been permeabilized,
cPrG-HCl inhibited both ATP- and pyrophosphate-dependent acidification of the vacuole. Application of cPrGHG1 to living internodal cells of Chara, induced a significant increase in vacuolar pH. The role of cPrG-HCl as
a tool for studying the physiological role of acidic vacuole is discussed.
tor of V-PPase has been reported.
To analyze the physiological role of the acidic vacuole in intact cells, it is necessary to stop the activity of
proton pumps. If only V-ATPase is playing a central role
for acidification of the vacuole, application of either
bafilomycin Al or concanamycin 4-B will be sufficient for
this purpose. If both V-ATPase and V-PPase are involved in acidification of the vacuole, activities of both
proton pumps must be inhibited.
A group of chemicals, prodigiosins, has been obtained from various bacteria resources (see for review, Geber
1975). Kataoka et al. (1995) reported that prodigiosin 25C, a member of prodigiosin, uncoupled V-ATPase and
inhibited acidification of lysosomes of rat liver. Kawauchi et al. (1997) reported that another member of
prodigiosins, cycloprodigiosin-hydrochloride (cPrG-HCl)
purified from marine bacterium Pseudoalteromonas denitrificans, also uncoupled V-ATPase of chromaffin granules. They stressed the advantage of using cPrG-HCl based
on the fact that it can be easily purified from the culture of
P. denitrificans and that it is very stable under various
physiological conditions. The possibility of cPrG-HCl as a
protonophore was excluded by the experiments showing
that cPrG-HCl did not affect membrane conductance of
phospholipid bilayers (Kawauchi et al. 1997). Whether
cPrG-HCl also inhibits vacuole acidification by plant VATPase, though suggested, has not been confirmed.
Key words: Characeae — Cycloprodigiosin — Proton
pump — Vacuole — V-ATPase — V-PPase.
The vacuole is the most prominent organelle in most
mature plant cells and occupies a large part of the total cell
volume. In addition to facilitating a drastic increase in cell
volume, the vacuole is an active intracellular compartment,
accumulating amino acids, sugars and secondary metabolites, and conducting hydrolysis activities. It is generally
accepted that maintenance of low pH is indispensable for
functioning of the vacuole. The plant tonoplast is equipped
with two different proton pumps, vacuolar-ATPase (VATPase) and vacuolar-pyrophosphatase (V-PPase) (Chanson et al. 1985, Marin 1985, Shimmen and MacRobbie
1987). The V-ATPase is inhibited by NO3~ and stimulated by Cl~ at the cytoplasmic side of the tonoplast (Churchill and Sze 1983, Rea and Poole 1985, Griffith et al. 1986,
Wang et al. 1986, Shimmen and MacRobbie 1987, Ward
and Sze 1992). Bafilomycin Al (Bowman et al. 1988) and
concanamycin 4-B (Drose et al. 1993) have been reported to
be specific inhibitors of V-ATPase. The V-PPase needs
K + at the cytosolic side of the tonoplast for its activity
(Chanson et al. 1985, Wang et al. 1986, Shimmen and
MacRobbie 1987). At present, however, no specific inhibiAbbreviations: ACS, artificial cell sap; APW, artificial pond
water; V-ATPase, vacuolar-ATPase; cPrG-HCl, cycloprodigiosin
hydrochloride; DMSO, dimethyl sulfoxide; EGTA, ethyleneglycol-bis-(/?-aminoethylether)/v',N,A''-./v'-tetraacei acid; pHv, pH in
the vacuole; V-PPase, vacuolar-pyrophosphatase; PPj, pyrophosphate; NR, neutral red.
1
Author for correspondence: Fax, 81-7915-8-0175; e-mail,
nakayasu@sci. himej i-tech .ac.jp
Internodal cells of Characeae have been a suitable
material for studying ion transport because of its large cell
size (Shimmen et al. 1994). In the present study, we analyzed the effect of cPrG-HCl on ATP- and pyrophosphate (PP;) -dependent acidification of the vacuole using
the internodal cells of Nitella after permeabilization of the
plasma membrane. We also analyzed the effect of cPrGHC1 on vacuolar pH of intact internodal cells of Chara.
Materials and Methods
Chara corallina and Nitella furcata were cultured in an airconditioned room (about 25 °C) as reported previously (Mimura
and Shimmen 1994). Internodal cells were isolated from neighboring cells and stored before use in artificial pond water (APW)
containing 0.1 mM each of CaCl2, KC1 and NaCl.
Analysis in permeabilized cells—Internodal cells of Nitella
furcata were used, since these cells were rather thinner and suitable for permeabilization. Young and growing internodal cells were
used, since old cells could not be permeabilized (Shimmen and
143
Cycloprodigiosin and vacuole acidification
144
MacRobbie 1987). It must be also noted that cells were permeabilized within about five hours after harvesting from the culture, since permeabilization becomes difficult with time after harvesting from culture.
Experiments were carried out according to Shimmen and
MacRobbie (1987). The vacuole was weakly stained with neutral
red (NR) by incubating internodal cells in APW supplemented
with 2 mM HEPES-NaOH (pH 7.5) and 30/iM NR. After cutting
both cell ends, the vacuoles were perfused with an artificial cell sap
(ACS) containing 120 mM KC1 and 10 mM CaCl2 (pH about 5.6,
unbuffered, 240 mOsM) by the method of Tazawa (1964). Complete replacement of natural cell sap with ACS was checked by
loss of the red color in the vacuole. By this perfusion, pHbuffering substances were removed from the vacuole. Two cell
fragments were prepared by ligation with polyester thread and
cutting the perfused cell; one fragment served as the control and
the other was used for the experiment to analyze inhibition
by cPrG-HCl. The plasma membrane of both cell fragments was
then permeabilized according to Shimmen and Tazawa (1983).
Cell fragments were first pretreated in an ice-cooled Mg(200)
medium containing 5 mM ethyleneglycol-bis-(/?-aminoethylether)/^N./V-AT-tetraacetic acid (EGTA), 6mM MgCl2, 30 mM
HEPES, 60 mM KC1, and 29 mM KOH (pH 7.5, about 200
mOsM) for 20-30 min to remove Ca 2+ from the cell surface. They
were then transferred into an ice-cooled Mg(340) medium containing 5 mM EGTA, 6 mM MgCl2, 30 mM HEPES, 60 mM KC1,
29 mM KOH, 140 mM sorbitol (pH7.5, about 340mOsM) and
incubated for 10 min to induce plasmolysis. By these treatments, the plasma membrane is permeabilized.
pump of the tonoplast, the permeabilized cell fragments were incubated in the Mg(340) medium supplemented with ATP (or
PPi) and 30/iM NR in the presence or absence of cPrG-HCl for
60-120 min in darkness at 24-26°C. The acidification of the vacuole was evaluated by the accumulation of NR in the vacuole,
which was determined from photographs. All experiments were
repeated at least five times and a typical result is shown in Fig. 1.
Analysis in intact cells—Internodal cells of Chara corallina
were used, since these cells were large enough to collect the cell sap
for pH measurement by the intracellular perfusion. After cells
were isolated from neighboring cells, they were kept in APW at
least overnight. Cells were incubated in APW supplemented with
1 mM HEPES (pH 7.2) in darkness at 24-26°C in the presence or
absence of cPrG-HCl. Before the measurement of vacuolar pH
(pHv), the velocity of cytoplasmic streaming was measured. Six
cells were used to measure pHv in every measurement and the
average value was shown with S.E. Measurement of pHv was
carried out according to Fujii et al. (1979). After cutting both cell
ends, the cell sap was pushed out and collected into a glass
capillary by slowly perfusing the vacuole with liquid paraffin.
Attention was paid to avoid contamination of the cytoplasm. The
collected vacuolar sap was blown out on Parafilm (American
National Can, Neenah, WI), and the pH was measured by a glass
pH electrode (Fuji Chemical Measurement Co. Ltd., model SE1700GC, Tokyo, Japan).
Permeabilization of the plasma membrane was checked by
observing the absence of the cytoplasmic streaming in the Mg(340)
medium lacking ATP at room temperature. To activate the proton
When permeabilized cell fragments were incubated in
the Mg(340) medium containing neither ATP nor PP;,
no accumulation of NR occurred as reported previously
-cPrG
Results
20 nM cPrG
A
B
Fig. 1 Effect of 20 nM cPrG-HCl on acidification of the vacuole in permeabilized cell fragments. Permeabilized cell fragments of
Nitella were incubated in the Mg(340) medium supplemented with either ATP or PPj in the absence (left) or presence (right) of 20 nM
cPrG-HCl. Acidification of the vacuole was evaluated by accumulation of NR. A: 1 mM ATP. B: 0.2 mM PP;. White lines running
obliquely in the longitudinal direction of the cell, more clearly visible in A (right) and B (right), are so-called indifferent zone, where the
chloroplast is absent. Bar represents 500 fim. See text for further explanation.
Cycloprodigiosin and vacuoie acidification
(Shimmen and MacRobbie 1987). In the presence of 1 mM
ATP in the Mg(340) medium, significant accumulation, of
NR was induced (Fig. 1A, left), indicating acidification of
the vacuoie. The effect of cPrG-HCl on ATP-dependent
NR accumulation was examined using another permeabilized cell fragment prepared from the same cell. The concentration of cPrG-HCl was adjusted at 20 nM, at which
ATP-dependent acidification of vesicles of chromaffin
granules is completely inhibited (Kawauchi et al. 1997).
ATP-dependent NR-accumulation was completely inhibited by 20 nM cPrG-HCl (Fig. 1A, right). In another series
of experiments, the same results were obtained (n=10).
Next, the effect of cPrG-HCl on PP r dependent accumulation of NR was examined. Takeshige and Tazawa
(1989) have reported the value of 193 //M for the PP; concentration in the cytoplasm of Chara. Therefore, the experiments were carried out using 0.2 mM PPj. In the presence of 0.2 mM PPj, significant accumulation of NR was
145
induced (Fig. IB, left). The NR accumulation induced by
0.2 mM PPj was completely inhibited by 20 nM cPrGHC1 (Fig. IB, right). When experiments were carried out
using 1 mM PP;, accumulation of NR was not completely
inhibited by 20 nM cPrG-HCl (data not shown).
About one hundred internodal cells were incubated in
APW (pH7.2) supplemented with cPrG-HCl. At each
time, the pH values of six cells were measured and the
average value is shown with SE. In Fig.2A, cells were incubated in APW supplemented with 0.05% (v/v) dimethylsulfoxide (DMSO) but without cPrG-HCl for a
control experiment. The medium containing cPrG-HCl at
50 nM, the highest concentration used in the present study
also contained 0.05% (v/v) DMSO. Before treatment, pHv
ranged between 4.9 and 5.0. During the 8-h incubation,
pHv slightly increased to be about 5.1. Treatment of cells
with 5 nM cPrG-HCl, caused a gradual increase in pHv to
about 5.4 (Fig.2B), and that with 20 nM cPrG-HCl gave
6.0 -•
6.0
••
Q.
co 5.5 - •
5.0
4.5
4
5
Time (h)
6.0
3
4
5
Time (h)
3
4
5
Time (h)
6.0 -•
••
Q.
to 5 . 5
" •
o
3
o
5.0
5.5 -•
5.0 - i
4.5
4.5
4
5
Time (h)
Fig. 2 Effect of cPrG-HCl on pHv in intact internodal cells of Chara. Cells were incubated in APW supplemented with cPrG-HCl at
various concentrations. The cell sap was collected by perfusing the vacuoie with liquid paraffin and its pH (pHv) was measured by a glass
pH electrode. A: APW containing 0.05% DMSO. B: 5 nM cPrG-HCl. C: 20 nM cPrG-HCl. D: 50 nM cPrG-HCl. Broken line in D: cells
were incubated in APW supplemented with 50 nM cPrG-HCl for 4-h and then they were transferred into freshly prepared same medium. Average values of six cells are shown with S.E.
Cycloprodigiosin and vacuole acidification
146
similar results, although the initial change was greater
(Fig. 2C). cPrG-HCl at 50 nM caused an increase in pHv to
about 5.8 and this pHv was maintained for 4-h. Then, pHv
began to decrease. After cells were incubated in APW
containing 50 nM cPrG-HCl for 4 h, they were transferred into a freshly prepared medium containing 50 nM
cPrG-HCl. After replacement of the cPrG-HCl medium,
pHv remained at 5.9-6.0 (Fig. 2D). Replacement of external medium containing 5 or 20 nM cPrG-HCl with fresh
cPrG medium of the same composition did not increase
pHv (data not shown). The cause of decrease in pHv after
a 4-h treatment with 50 nM cPrG-HCl remains to be
solved.
Also, cPrG-HCl had no significant effect on the velocity of cytoplasmic streaming at any concentration of
cPrG-HCl tested. Fig. 3 shows the results obtained with 50
nM cPrG-HCl. This indicates the absence of any drastic
changes of cytoplasmic pH and ATP level, which strongly
affect cytoplasmic streaming (Shimmen 1978, Tazawa and
Shimmen 1982).
Discussion
To examine the physiological role of the acidic vacuole, the experiments to increase pHv by inhibiting the activity of proton pumps is one of the most promising
strategies. The novel inhibitors bafilomycin Al (Bowman
et al. 1988) and concanamycin 4-B (Drose et al. 1993)
have been applied to various plants to elucidate the physiological role of V-ATPase (Okazaki et al. 1992, Tazawa et
al. 1995, Romani et al. 1996, Muller et al. 1996, Brauer et
al. 1997).
In addition to V-ATPase, it became evident that VPPase also plays an important role in manifestation of
3
4
5
Time (h)
Fig. 3 Effects of 50 nM cPrG-HCl on the velocity of cytoplamic streaming. Cells were incubated in APW supplemented
with 50 nM cPrG-HCl and the velocity of cytoplasmic streaming
was measured at various time. Broken line: cells were incubated in
APW supplemented with 50 nM cPrG-HCl for 4-h and then they
were transferred into the same freshly prepared medium. Average
values of six cells are shown with S.E.
vacuole functions (Johannes and Felle 1990, Carystinos et
al. 1995, Darley et al. 1995, Macri et al. 1995, Shiratake et
al. 1997, Nakanishi and Maeshima 1998). Neither bafilomycin Al nor concanamycin 4-B inhibited PPase (Okazaki et al. 1992, Matsuoka et al. 1997). Although the activity of PPase is inhibited by depleting K + from the
cytoplasmic side of the tonoplast, such treatment can be
applied only to tonoplast vesicles or permeabilized cells
(Chanson et al. 1985, Wang et al. 1986, Shimmen and
MacRobbie 1987), and not to intact cells.
Okazaki et al. (1992) applied bafilomycin Al to internodal cells of Chara. Bafilomycin Al at 0.1 /uM did not
induce an increase in pHv in intact cells. However, bafilomycin Al induced an increase in pHv in internodal cells
whose cell sap had been replaced with an artificial cell sap
lacking buffering capacity. They reported that PP r dependent H + -pumping alone was insufficient for the pH
regulation of the vacuole, based on the fact that bafilomycin Al applied into the vacuole induced an increase in
pHv. They also suggested the possible involvement of PPase in the acidification of the vacuole, based on the fact
that bafilomycin Al could not completely inhibit vacuole
acidification. Tazawa et al. (1995) applied concanamycin
4-B, to internodal cells of Chara. Concanamycin 4-B at 1
//M applied to the cell exterior of intact internodal cells
induced a slight increase in pHv, but not at 0.1 yuM. An
increase in the concentration of concanamycin 4-B up to 10
//M did not induce further increase in pHv. Katsuhara et al.
(1989) observed a decrease in cytoplasmic pH and increase
in pHv upon NaCl stress in Nitellopsis obtusa. Based on
the facts that the Na + concentration in the cytoplasm increases upon salt stress (Katsuhara and Tazawa 1986) and
that V-PPase is inhibited by Na + (Takeshige and Hager
1988), it was concluded that PPase is functioning and essential in keeping the H + -gradient across the tonoplast in
addition to V-ATPase. On the other hand, Tazawa et al.
(1995) concluded that the pHv regulation under salt stress
is achieved exclusively by the V-ATPase, based on the fact
that concanamycin 4-B completely inhibited reacidification of the vacuole upon NaCl stress in Chara. Matsuoka et
al. (1997) reported that neither bafilomycin Al nor concanamycin 4-B induced a change of pHv in tobacco cultured cells. Thus, it seems that contribution of two proton
pumps in regulation of pHv is greatly variable among cells
and physiological conditions. At any rate, the artificial increase in pHv is one of the most promising ways to analyze the physiological role of the acidic vacuole.
The present study showed that cPrG-HCl at 20 nM
completely inhibited both ATP- and PP,-dependent acidification of the vacuole in permeabilized Nitella cells. We
also found that cPrG-HCl at 50 nM induced a significant
increase in pHv in intact Chara cells. The pHv remained at
around 5.8 even in the presence of 50 nM cPrG-HCl. Involvement of buffering capacity and biochemical pH stat in
Cycloprodigiosin and vacuole acidification
pHv regulation in intact cells must be considered (Smith
and Raven 1979).
To establish cPrG-HCl as a useful tool for studying
the physiological role of acidic vacuoles, the following
must be elucidated. Kawauchi et al. (1997) showed that
cPrG-HCl inhibited acidification but not ATP hydrolysis
activity in chromaffin granules. This must be examined in
plant V-ATPase and V-PPase. Such experiments are possible only in tonoplast vesicles. We are planning experiments to analyze the effects of cPrG-HCl on hydrolysis
and acidification in vesicles of higher plants. Sato et al.
(1998) reported that prodigiosin, metacloprodigiosin and
prodigiosin 25-C uncoupled the activity of proton pumps
as a H + /C1~ symporter. This possibility must be also examined in cPrG-HCl.
Since the velocity of cytoplasmic streaming was not
affected by cPrG-HCl even at 50 nM, the drastic change in
the chemical compositions in the cytoplasm, such as
ATP concentration and pH, may not have been caused by
the treatment with cPrG-HCl. The effect of cPrG-HCl on
F^o-ATPase of mitochondria and chloroplasts, the proton
pump of the plasma membrane must be examined to establish cPrG-HCl as a suitable probe for analyzing the
physiological role of acid vacuoles in plant cells.
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(Received August 26, 1998; Accepted November 17, 1998)