Journal of Experimental Botany, Vol. 55, No. 408, pp. 2505–2512, December 2004
doi:10.1093/jxb/erh256 Advance Access publication 10 September, 2004
RESEARCH PAPER
Ion flux interaction with cytoplasmic streaming in
branchlets of Chara australis
Olga Babourina*, Konstantin Voltchanskii and Ian Newman
School of Mathematics and Physics, University of Tasmania, GPO Box 252-21, Hobart, Australia 7001
Received 22 April 2004; Accepted 16 July 2004
Abstract
Both parts of the actin–myosin complex involved in
cytoplasmic streaming could be regulated by mineral
ions. The main goal of this study was to find a relationship between cyclosis and ion transport across the cell
wall and plasma membrane. The transport of K1 and
Ca21 along pH bands in Chara branchlet internodal
cells was characterized by using the MIFE system for
non-invasive microelectrode measurement of ion fluxes.
Branchlets formed acidic and alkaline bands with the
pH ranging from 5 to 8. Different pH patterns were
observed for different sides of the branchlets. Sides
with cyclosis streaming acropetally generally showed
greater variation in the profiles of pH and H1 fluxes.
Although a high correlation was not found between pH
bands and Ca21 or K1 fluxes, there was a positive correlation between Ca21 and K1 fluxes themselves for both
sides of the branchlets. Application of cytochalasin D,
an inhibitor of cyclosis, had no immediate effect on pH
and ion fluxes, however, the time of cyclosis cessation
corresponded with a dramatic change in Ca21 and K1
fluxes; pH profiles and H1 fluxes were affected within
2 h. The evidence suggests that, in Chara branchlets, pH
band formation and Gd31-insensitive Ca21 transport
systems are linked to the cyclosis machinery: (i) the pH
band amplitude for the acropetally streaming side was
larger than that for the basipetally streaming side; (ii)
cessation of cytoplasmic streaming after cytochalasin D
application resulted in changed pH banding profiles and
H1, Ca21 and K1 fluxes; and (iii) the application of GdCl3
or incubation in GdCl3 solutions did not lead to the
cessation of cytoplasmic streaming, although external
Ca21 fluxes changed.
Key words: Calcium, cyclosis, cytochalasin D, pH, potassium,
proton.
Introduction
Most eukaryotic cells show cytoplasmic streaming. The
motive force for streaming is generated by the sliding
movement of myosin with bound vesicles, along the bundle
of actin filaments, using energy derived from ATP hydrolysis (Alberts et al., 1994; Shimmen and Yokota, 1994).
Chara cells show very fast endoplasmatic streaming,
probably because of the properties of their myosins, the
fastest (60 lm sÿ1) of all plant myosins observed so far
(Morimatsu et al., 2000). Cytoplasmic streaming is obviously linked with the metabolic production of ATP for
myosin movement and for actin polymerization. The link
between cytoplasmic streaming and ion transport is less
apparent. Reviewing the literature, Hope and Walker
(1975) noticed that a temporary halt in cyclosis was caused
by all stimuli that produce an action potential (mechanical
shock, bending, and a sudden drop in temperature). They
suggested that this could be linked with Ca2+ influx into
the cytoplasm, however, at that time there was no direct
evidence of a link between [Ca2+]cyt and cyclosis. It has
since been confirmed that [Ca2+]cyt is increased during
action potentials (Williamson and Ashley, 1982; Hayashi
and Takagi, 2003).
Because six binding sites for calmodulin have been
found in Chara myosins, it is plausible that Ca2+ can participate in regulating myosin action (Morimatsu et al., 2000).
However, there is some controversy over Ca2+ involvement
in myosin action. In an in vitro motility assay, Chara
myosin did not show Ca2+ sensitivity (Higashi-Fujime
et al., 1995), but in vitro motile activity of myosin isolated
* Present address and to whom correspondence should be sent: School of Earth and Geographical Sciences, M087, University of Western Australia, 35
Stirling Highway, Crawley, WA 6009, Australia. Fax: +61 8 9380 1050. E-mail: [email protected]
Abbreviation: CD, cytochalasin D.
Journal of Experimental Botany, Vol. 55, No. 408, ª Society for Experimental Biology 2004; all rights reserved
2506 Babourina et al.
from the pollen tubes of lily was inhibited by Ca2+ (Yokota
et al., 1999).
Changes in [Ca2+]cyt are not the only events observed
during action potentials that might cause cytoplasmic
streaming to stop. Activation of Clÿ and K+ channels also
contribute to the action potential (Oda, 1976; Hayama et al.,
1979; Shimmen et al., 1994). Hence, temporary cyclosis
cessation is linked with changes in cytoplasmic concentrations of at least three mineral ions: Ca2+, K+, and Clÿ.
Involvement of K+ and Clÿ in the action potential could be
of secondary origin as a consequence of plasma membrane
depolarization or increased [Ca2+]cyt. However, there is
some indication that K+ could be directly involved in the
regulation of cytoplasmic streaming. Actin filaments need to
be constantly polymerized from their plus end at a certain
rate; any disruption in polymerization causes disruption in
their functions, including cyclosis. Polymerization of pure
actin in vitro requires ATP, as well as both monovalent and
divalent cations, usually K+ and Mg2+ (Alberts et al., 1994).
Thus, both parts of the actin–myosin complex involved in
cytoplasmic streaming could be regulated by mineral ions,
whose transport would therefore change during the cessation
of cyclosis. The main goal of this study was to find a relationship between cyclosis and ion transport across the cell wall
and plasma membrane. Light microscopy and the MIFE
system for non-invasive microelectrode measurement of ion
fluxes were used: (i) to characterize the transport of H+, K+,
and Ca2+ along acid and alkaline bands in branchlet cells and
(ii) to compare the ion transport of the branchlets and the
nodes before and after inhibition of cyclosis by cytochalasin D
(CD) and inhibition of Ca2+ influx by GdCl3 treatment.
Ion flux measurements
Ion fluxes were measured non-invasively using the MIFE system
(University of Tasmania, Hobart, Australia) generally as described
by Newman (2001). The ion-selective microelectrodes were backfilled
by 0.15 mM NaCl and 0.4 mM KH2PO4 (adjusted to pH 6.0 using
NaOH) for hydrogen, 0.5 M NaCl for sodium, and 0.5 M KCl for
potassium. The electrode tips were filled with commercial ionophore
cocktails (hydrogen 95297; potassium 60031; calcium 21048, Fluka).
The electrodes were calibrated in a set of standards (pH from 4.6 to
7.8; Ca2+ from 0.1 to 10 mM; K+ from 1 to 100 mM). Electrodes
with responses less than 50 mV per decade for monovalent ions and
25 mV per decade for calcium were discarded.
Experimental procedure
The Chara cells were measured in the same Petri dishes placed on an
inverted microscope. During measurements the cells were immobilized by a small piece of glass, which was placed on the cell wall
remaining from the neighbouring internodal cell.
Electrodes were lowered into the solution and their tips were
positioned near the cell in the plane of its optical section. They were
moved horizontally along a cell radius between 30 lm and 60 lm
from its surface using a WR88 micromanipulator (Narishige, Japan)
in a square-wave manner with a 10 s cycle. Experiments were
performed at 20–22 8C, under normal laboratory lighting. Fluxes of
the three specific ions were measured simultaneously.
In mapping experiments each point, at 0.2–0.5 mm separations
along the cell axis, was measured for 2–3 min. Since cells varied in
size, about 761 mm, they were normalized to 7 mm, recalculating
proportionally the other distances along the cell. For each cell, mean
pH and fluxes over these times at each point were calculated and
plotted. For each cell, correlations between pH and fluxes along the
cell were calculated from those mean values (n=18–31). Means of
correlation coefficients from 12 cells are shown in Table 1, with
standard errors of those means. Mapping after CD and Gd3+ treatments was made at least 2 h after the reagent addition.
In experiments with CD (bath concentration 25 lM) and GdCl3
(500 lM), stock solutions were added to the Petri dish in several spots
Materials and methods
Plant material
Chara australis R. Br. cells were taken from an established indoor
culture grown in a plastic tank. Individual branchlet cells with
attached nodes or isolated branchlet nodes, as indicated in Fig. 1,
were cut 4–6 h before the experiments. They were placed horizontally
in Petri dishes with 2–3 ml artificial pond water (APW: 0.1 mM KCl,
0.1 mM CaCl2, 0.5 mM NaCl, unbuffered pH 5.5). Chara branchlet
cells were chosen for use because the whole cell can be viewed in
one microscope field and the cessation of cytoplasmic streaming was
considered as an event when it stopped in the whole cell, not only in
the location of electrodes.
Fig. 1. Schematic diagram of Chara australis branchlet cells. (A) The
second whorl from the top of the plant. The branchlet cell that was used in
experiments is circled. (B) The side of the branchlet where cyclosis went
acropetally is designated side A; where it went basipetally, side B.
Table 1. Correlation between H+, K+, and Ca2+ fluxes and pH for both sides of Chara branchlets
Correlation coefficients were calculated for each cell separately. Statistics are means of coefficients from 12 cells 6SE. Levels of significance for the
correlation coefficient are indicated by (*) for 5% and by (**) for 1%.
Side A
pH
H+
K+
Ca2+
Side B
pH
H+
1
0.6660.06*
0.1760.07
0.1360.11
1
0.1660.09
0.2260.09
K+
1
0.7960.05**
Ca2+
pH
H+
K+
Ca2+
1
1
0.8260.03**
0.4960.04
0.5460.07
1
0.5860.04*
0.7060.03*
1
0.6760.04*
1
Interaction of ion fluxes and cyclosis 2507
in a circle close to the edge. The bath solution was mixed by sucking
and expelling it from a 1 ml pipette several times. The time required
for reagent addition, mixing, and establishing the diffusion gradients
was discarded from the analysis and appears as a gap in Figs 3 and 5
of the Results. The CD stock solution was freshly made every day
before the experiments by dissolving CD in DMSO to give a final
concentration of DMSO of 0.2% (v/v) in the Petri dish. The inhibition
of cyclosis was assessed visually under the microscope. No effect of
0.2% DMSO on cyclosis and ion fluxes was observed in control
experiments.
Cytoplasmic streaming was assessed under the microscope, with
the chloroplasts in focus to measure the streaming rate at the site of
the generation of the motive force (Staves, 1995), by counting the
time required for small particles to travel 200 lm.
Results
Ion flux mapping of Chara branchlets and their nodal
cells
As expected, branchlets formed alkaline and acidic bands
whose pH ranged from 5 to 8. The pH profile and the
K+, Ca2+, and H+ flux profiles along each side of three
representative Chara branchlet cells are presented in Fig. 2.
Different pH and ion flux patterns were observed for
different sides of the branchlets. Sides with cytoplasm
streaming acropetally (side A) had the greater variation in
pH and H+ fluxes. The mean amplitude of this variation, for
Fig. 2. Net pH profiles (acid upwards) and ion fluxes (influx positive) along each side in three representative branchlet cells. ‘N’ indicates the basal node.
(A, B) pH profiles for sides A and B; (C, D) H+ fluxes; (E, F) Ca2+ fluxes; (G, H) K+ fluxes. Initial pH or flux values are shown at the left of each side A
trace. Initial values for side B are similar.
2508 Babourina et al.
pH and each flux in the most prominent pH band of side A,
is shown in Table 2 (control).
Generally, H+ fluxes were consistent with H+ extrusion being the cause of acidic banding: at regions of
lower pH greater H+ efflux was observed, while for
alkaline areas the efflux was smaller or even shifted to
influx. Thus, the correlation between pH and H+ fluxes
was high: 0.66 for side A (P<0.05) and 0.82 for side B
(P<0.01) (Table 1). For acropetal streaming (Table 1,
side A) there was only small, insignificant correlation
between pH or H+ flux and either K+ or Ca2+ fluxes. For
basipetal streaming (Table 1, side B) those correlations
were much higher and positive: between H+ and K+ or
Ca2+, correlation coefficients were both significant
(P<0.05). For both sides there was significant correlation
between K+ and Ca2+ fluxes: 0.67 for side B (P<0.05)
and 0.79 for side A (P<0.01).
The branchlet cell basal node showed two differences
from the cell. (i) The node had relatively stable pH in the
range 6.1 to 6.5, midway between the banding extremes for
the cell. (ii) The node had a higher Ca2+ influx (or smaller
efflux) than the adjacent area of the attached branchlet
cell for side A (Fig. 2). The side A mean of the Ca2+ flux
Table 2. Magnitude of variation in pH and each ion flux in the
strongest band of side A in a branchlet cell before (control) and
2 h after application of CD or Gd3+, or CD with Gd3+
pretreatment, for the same band
The strongest band was chosen from its pH/H+ amplitude. Flux units,
nmol mÿ2 sÿ1. Means 6SEM (n=4–12). Levels of significance for flux
difference before and after treatments are indicated by ** for 1% and by
**** for 0.01%.
pH
H+
K+
Ca2+
Control
+CD
+Gd3+
+CD after Gd3+
0.6360.04
48.366.1
43.567.6
33.763.9
0.0860.01****
0.860.09****
8.660.8****
5.660.2****
0.5460.1
33.766.7**
1261.3****
4.560.7****
0.0360.01****
5.260.2****
2166.0****
3.660.2****
difference between the node and its adjacent cell is 3364
nmol mÿ2 sÿ1 (SE, n=6). For K+ flux, this mean difference
is 5610 nmol mÿ2 sÿ1 (SE, n=6).
Correspondence between ion fluxes and cytoplasmic
streaming
The application of 25 lM CD, which prevents actin from
polymerizing, immediately slowed cyclosis, with inhibition
complete by about 15 min (Fig. 3), in agreement with
Collings et al. (1996).
H+ fluxes, measured at a location about 2 mm from
the basal node, demonstrated a multiphasic response with
an initial slight shift towards influx, subsequently returning
almost to initial values (Fig. 3). After cyclosis stopped, H+
fluxes tended to influx. Further exposure for 2 h or more led
to a significant reduction of H+ and pH banding patterns
(Fig. 4A; Table 2).
Net K+ fluxes, also measured about 2 mm from the basal
node, showed no significant change until an abrupt shift
towards efflux happened a few minutes after cyclosis ceased
(Fig. 3). Ca2+ fluxes demonstrated a slight initial efflux
(opposite to the H+ flux changes) in the first 5-10 min after CD
application, followed by a trend to recover the initial values.
However, similar to K+ fluxes 5–10 min after the cessation of
cyclosis in the observed area, net Ca2+ flux changed towards
efflux. In absolute values, 2 h of cell exposure to 25 lM CD
resulted in a significant reduction in magnitudes of K+ and
Ca2+ flux variation within the bands (Table 2), with direction
changed from influx to efflux by 2962 nmol mÿ2 sÿ1 for K+
and 2261 for Ca2+ (SE, n=3) (Fig. 4B).
To help identify the process responsible for changes in
pH banding, cytoplasmic streaming cessation or changes
in ion transport itself, the Ca2+ channel blocker Gd3+ was
used. Application of 500 lM Gd3+ induced enormous
transient efflux of Ca2+ from both the node and its branchlet
(Fig. 5). Following the transient (lasting 15 min for nodes
and 5 min for branchlet cells) a small net Ca2+ efflux
Fig. 3. A representative experiment (out of 12) showing the effects of the addition of 25 lM cytochalasin D into the medium at 5 min (arrowed).
Cytoplasmic streaming was recorded in the area that could be observed under the microscope close to the electrodes. Electrodes were positioned at 2 mm
from the basal node of side A. Initial cyclosis speed was about 60 lm sÿ1. Cyclosis stopped about 15 min after CD application.
Interaction of ion fluxes and cyclosis 2509
Fig. 4. (A, B) Representative profiles of ion fluxes and pH along side A before (open symbols) and after (filled symbols) 2 h incubation of one cell in
25 lM CD. (C, D) Representative profiles of ion fluxes and pH along side A initially (open symbols), after 2 h incubation in Gd3+ (black symbols) and
after subsequent 2 h incubation in CD (grey symbols).
continued. Two hours after Gd3+ application (n=4), Ca2+
fluxes had changed towards efflux in zones where initially
there was influx, or to a bigger efflux where there was
already efflux (Fig. 4D). The mean change was 2763 nmol
mÿ2 sÿ1 (SE, n=4) for the whole cell. Gd3+ application also
caused a transient H+ efflux of similar duration to the Ca2+
transient (Fig. 5). This observation is in good agreement
with earlier findings made on internodal cells of Chara
corallina, where H+ currents were transiently inhibited by
Gd3+ and La3+ (McConnaughey and Falk, 1991). However,
2 h pretreatment with 500 lM GdCl3 had little effect on
H+ fluxes or pH (Fig. 4C) and on the amplitudes of H+ and
pH profiles (Table 2). The immediate (Fig. 5) and prolonged
(Fig. 4D) effect of Gd3+ on K+ fluxes was negligible,
although with some small initial transient efflux from the
node. During and after GdCl3 application, cyclosis was
observed to be unaffected (data not shown).
The massive transient ion efflux, with a time-course of
around 15 min, is consistent with the trivalent Gd3+
displacing Ca2+ (and to a lesser extent H+ and K+) from
the Donnan system of the cell walls (Ryan et al., 1992). The
longer term flux changes are not affected by this exchange.
For cells pretreated for 2 h in 500 lM GdCl3, a subsequent 2 h addition of 25 lM CD led to the cessation of
cytoplasmic streaming (data not shown) and to a significant
reduction in the magnitude of H+ fluxes (Fig. 4C). This
treatment also reduced the amplitudes of the pH and H+
profiles (Table 2; Fig. 4C) in a way similar to the effect of
CD on cells without Gd3+.
To summarize the results presented in Fig. 4A and C, and
in Table 2, for prolonged treatments with CD and Gd3+, H+
fluxes and pH are mainly influenced by CD treatments
rather than by the application of the Ca2+ channel blocker
Gd3+. Ca2+ and K+ fluxes were affected by each pretreatment. These observations, with the correlation levels presented in Table 1 for cells without any treatments, indicate
that K+ and Ca2+ transport in Chara cells is independent of
H+ transport and pH band formation.
Discussion
Ion fluxes along alkaline and acidic bands
It has been known for 30 years that internodal cells of
Chara form acid and alkaline bands during illumination
(Lucas and Smith, 1973). Despite the pH band phenomenon
having been confirmed in numerous studies, the mechanism
of its formation and its function are still unclear. On the
2510 Babourina et al.
Fig. 5. Transient changes in the net ion fluxes (efflux negative) measured from a representative node and at an alkaline band in its branchlet cell in
response to application of 500 lM GdCl3.
other hand, transport of many ions at the boundary of the
Chara internodal cell with the bathing solution has been
characterized using different techniques. Radioisotopes
were used in studies on Na+, Clÿ, and Ca2+ fluxes (Hope
and Walker, 1975; Findlay et al., 1969; Sanders, 1980;
MacRobbie and Banfield, 1988; Hoffmann et al., 1989;
Whittington and Smith, 1992; Whittington and Bisson,
1994). K+ and Clÿ channels, inward and outward rectifiers
were characterized in Chara using voltage and patch
clamping techniques (Smith, 1984; Beilby, 1985; Tyerman
et al., 1986; Coleman 1986; Kourie and Findlay, 1990;
Homann and Thiel, 1994; McCulloch et al., 1997). Ion
specific microelectrodes for Ca2+ were used to characterize
Ca2+/H+ exchange in the wall during algal calcification
(McConnaughey, 1991; McConnaughey and Falk, 1991),
and during progressively increased external concentrations
of K+ and H+ (Ryan et al., 1992). However, with current
knowledge, the present work is the first attempt to characterize the contribution of different ions in the overall electrical currents along the pH profiles in Chara.
From these results it is clear that OHÿ/H+ fluxes
contributed significantly in the formation of alkaline and
acidic bands, confirming theoretical calculations made by
Lucas et al. (1977). Despite an emerging literature showing
the regulation of K+ transport by external or internal pH in
plants (Hoth et al., 1997; Gaymard et al., 1998; Lacombe
et al., 2000), these results link K+ transport with Ca2+ rather
than with pH: for both sides K+ and Ca2+ fluxes are
significantly correlated (Table 1). In fact, interdependence
between K+ and Ca2+ transport for Chara has been described previously. Removal of Ca2+ from the medium
results in the depolarization of the Chara internodal cell and
an increase in membrane conductance (Gm). The increase in
conductance was thought to be associated with an increase
in K+ conductance, as judged by Ca2+ effects on the K+
dependence of clamp current (Bisson, 1984). First of all,
this interdependence could be explained by a Ca2+ effect on
K+ transport: strong Ca2+ dependence of K+ channels has
been shown for many higher plants and algae (Spalding
et al., 1992; Plieth et al., 1998; Bauer et al., 1998).
Conversely, it is possible that Ca2+ transport is regulated by
K+ via changes in Em: voltage regulation of Ca2+ channels
has been demonstrated in many studies (Katsuhara and
Tazawa, 1992; Shimmen, 1997; Reid et al., 1997).
Interaction of ion transport with cytoplasmic streaming
Ca2+ and K+ transport: These results indicate that Ca2+ and
K+ transport is closely linked with cytoplasmic streaming.
Interaction of ion fluxes and cyclosis 2511
The time at which cyclosis stops in the whole cell coincides
with increased Ca2+ influx and, probably as a consequence,
the change in K+ fluxes. Dependence of ion transport on the
cytoplasmic streaming rate has been described previously.
In studies on ion transport across the node from one internodal cell to another, it was found that rate of intercellular
Clÿ and Rb+ transport is highly dependent on the rate of
cytoplasmic streaming (Bostrom and Walker, 1976; Ding
and Tazawa, 1989). It has also been demonstrated that
cytoplasmic streaming depends on [Ca2+]cyt, with high
concentrations of [Ca2+]cyt causing inhibition (Tominaga
and Tazawa, 1981; Williamson and Ashley, 1982).
H+ fluxes and pH band formation: Further evidence for the
interaction of cytoplasmic streaming with ion fluxes is the
observed asymmetrical distribution of pH bands at the sides of
the branchlet cell having opposite directions of cytoplasmic
streaming, since cells were placed horizontally so gravitropically induced streaming polarity was abolished (Staves,
1997). An amplitude difference between the two sides of
the internodal cell was also detected by Lucas and Nuccitelli
(1980) and Bulychev et al. (2001). Unfortunately, in both
papers the authors did not indicate the direction of the
cyclosis, which does not allow the results to be compared.
The literature provides some evidence on the link between pH bands and the cytoskeleton, both microtubules
and the actin–myosin complex, responsible for cytoplasmic
streaming. Exposure of Chara cells to vinblastine, colchicine, or oryzalin caused a reduction in the extracellular current pattern and a shifting of it (Fisahn and Lucas, 1990).
Because these agents all affect tubulin, the authors suggested that microtubules could be responsible for orchestrating the transmembrane currents. Further microscopical
studies on microtubule alignments along pH bands established that microtubule number and alignment change between acidic and alkaline bands in Chara cells (Wasteneys
and Williamson, 1992). However, these authors found that
pH band formation was not prevented when microtubules
were depolymerized with oryzalin, demonstrating that microtubules are not necessary for pH bands to develop in
internodes. They concluded that some mechanisms for
microtubule organization respond to the localized differences in membrane properties, rather than being responsible
themselves for pH banding formation.
Few papers have described the link between the functioning of the actin–myosin complex and the formation of pH
bands: (i) cyclosis inhibition transforms the band pattern to
one of numerous small discs (Lucas and Dainty, 1977); (ii)
slowing cytoplasmic streaming corresponds with narrower
and more numerous bands (Yao et al., 1992); (iii) treatment
of cells with cytochalasins, inhibitors of cytoplasmic streaming, caused the banding pattern to disappear (Bulychev et al.,
2001). The results of this study support the last observations.
The results of this study lead to three conclusions: (i) It is
proposed that pH band formation and Gd3+-insensitive Ca2+
transport systems in Chara branchlets are linked with the
cyclosis machinery: (a) sides with different directions of
cytoplasmic streaming produce patterns with different pH
amplitude range; (b) application and incubation in the GdCl3
solutions did not lead to the cessation of cytoplasmic
streaming, although external Ca2+ fluxes changed. (ii) K+
and Ca2+ transport in Chara cells are not strongly linked with
H+ transport: pre-treatment with Gd3+ led to changed transport properties of K+ and Ca2+, whereas H+ transport
remained almost unaffected. (iii) There is a link between
ion transport and cytoplasmic streaming: cessation of cytoplasmic streaming, some time after CD application, resulted
in changed H+, Ca2+, and K+ fluxes and pH banding.
Acknowledgement
This work was supported by an Australian Research Council grant to
Dr Newman (A00105708).
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