Plant Cell Physiol. 30(5): 739-748 (1989)
JSPP © 1989
Influence of Cytoplasmic Streaming and Turgor Pressure Gradient
on the Transnodal Transport of Rubidium and Electrical
Conductance in Chara corallina
Da-Qiao Ding and Masashi Tazawa
Department of Botany, Faculty of Science, University of Tokyo, Hongo, Tokyo, 113 Japan
Key words: Chara corallina — Cytoplasmic streaming — Electrical resistance — Plasmodesmata —
Transnodal transport — Turgor pressure gradient.
Intercellular communication is one of the fundamental processes in most multicellular organisms. Corresponding to gap junctions in animal cells, there are thin
cytoplasmic channels called plasmodesmata between adjaAbbreviations: APW, artificial pond water; CB, CE,
cytochalasin B and E; DMSO, dimethylsulfoxide; EDTA, ethylenediaminetetraacetic acid; HEPES, W-2-hydroxyethyIpiperazine-W-2-hydroxypropane-3-ethane-sulfonic acid; MES, 2-(JVmorpholino)ethanesulfonic acid.
cent plant cells. Intercellular chemical communication in
plant cells is thought to occur mainly through plasmodesmata (Gunning and Robards 1976).
Plasmodesmata are thin cytoplasmic tubes through
the cell wall between two neighboring cells. They have a
diameter of about 50-100 nm (Overall et al. 1982), and they
usually contain a desmotubule. Desmotubules are formed
from endoplasmic reticulum when cell division occurs
(Hepler 1982). Materials having a molecular weight of less
than 665 dalton can pass freely through the plasmodesmata
739
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In order to study the transnodal transport of Rb + in internodal cells of Chara corallina, a
low-temperature loading system was established to separate the loading process from the transport process. Tandem cells, consisting of internode-node-internode, were isolated from algal
plants. Treatment of a single internode with 100 mM RbCl at 5°C for 30 min caused an accumulation of 43 mM Rb + in the cytoplasm of this cell ( = source cell), but no Rb + was found in the
other internode ( = sink cell) of the tandem cells. In 40 min after a return to 25°C, about 12% of
the Rb + loaded in the source cell was transported into the sink cell. The apparent transnodal
permeability of Rb + was calculated to be 4.6 x 10~7 m s " 1 . Under the assumption that the total
cross-sectional area of plasmodesmata occupies 10% of the nodal area, the diffusion coefficient of
RbCl through plasmodesmata was calculated to be 2.3 x 10"" m 2 s ~ ' which is about \% of the
free diffusion coefficient in water (2x 10~ 9 m 2 s~').
The transnodal transport of Rb + was intimately correlated with the rate of cytoplasmic
streaming. The rate of streaming in both the source and sink cells was varied either by treating
the cells with cytochalasin B (CB) or by lowering the temperature. The transport rate correlated
with the streaming rate irrespective of the method used. Since the level of ATP was not influenced by CB or low temperature, the transnodal transport is assumed to be the result of passive diffusion process through plasmodesmata.
A turgor pressure gradient across the node decreased both the nodal electrical conductance
and the transnodal transport of Rb + . By contrast, the exposure of both internodal cells to a solution of sorbitol had no effect on either of them. A turgor pressure gradient of 240 mOsm decreased the transport of Rb + in the first hour to 3% of the control, while it decreased the nodal
conductance to about 50%. The increase in the electrical resistance occurred on the junction side
between the node and the internode that was treated with sorbitol. Cytochalasin E had no effect
on the nodal electrical resistance. It is assumed that plasmodesmata are equipped with a valvelike mechanism which is sensitive to the gradient of turgor pressure across the node and is not
regulated by an actomyosin system.
D. Q. Ding and M. Tazawa
740
Materials and Methods
Plant material—Chara corallina was cultured in
plastic buckets with tap water that contained soil extract
and rotten leaves. The buckets were placed in an air-conditioned room of 25 ± 1 °C and illuminated with fluorescent
lamps (20 W x 2) for about 16 hours a day.
At least two days before each experiment, nodal complexes were dissected from the main axis of plants. Each
nodal complex consisted of one node at the center and two
adjacent internodal cells. Leaf cells were trimmed away
from the node. Cells were treated for 3 hours with an
acidic solution of APW, which contained 10 mM MES and
0.1 mM each of KC1, NaCl, CaCl2 (pH4.5) to remove the
deposits of calcium carbonate from the cell walls. Cells of
more than 6 cm in length were ligated with strips of polyester thread at loci 6 cm away from the node. By cutting
off the distal cell fragments, we obtained a nodal complex
with one internodal cell of fixed length (6 cm) on either side
of the node (Fig. 1). Nodal complexes prepared in this
way were incubated at 25°C in APW that contained 0.1 mM
each of KG, NaCl and CaCl2, in an air-conditioned room.
Transnodal transport experiments—In experiments to
measure the transnodal transport of Rb + , a nodal complex
was set in a Plexiglas chamber as shown in Figure 1. This
chamber has three compartments (A, N, B). The internodal and nodal cells were separated by two partition walls
that had grooves of 0.5 cm in depth. The internodes were
fitted into the grooves with silicone grease. The central
nodal part consists of the node and adjacent small portions
of internodes. The length and diameter of the two internodes were measured. The cells were then incubated in
APW for 1 hour before loading to minimize the effect of
handling. Rb + loading was carried out by addition of
100 mM RbCl to chamber A. To ensure isoosmotic conditions between the two cells, a 180 mM solution of sorbitol
was added to compartment B. Loading was allowed to
proceed for half an hour at 5°C. We call the internode
that was loaded with Rb + the source cell and the internode
that was not loaded the sink cell.
After loading, RbCl and sorbitol were washed away
with a solution of 1 mM CaCl2 and 1 mM NaCl. Washing
was repeated three times at room temperature. Then all
the compartments were filled with APW to initiate transport. Normally the experiment was performed for 1 to
3 hours at 25°C in an air-conditioned room. In the lowtemperature experiments, the chamber was placed in an incubator that was adjusted to 5, 10, 15, or 20°C. The cells
were illuminated with two 20-W fluorescent lamps. When
CB (dissolved in DMSO) was used to control the streaming
rate, 10-30 fig/ml CB was added at the beginning of the
loading period. After Rb + loading and subsequent washing, the chamber was filled with a CB-APW solution of the
same concentration as was used in the previous loading period. The rate of cytoplasmic streaming was monitored
s o u r c e
n k
Fig. 1 Experimental system for measuring transport of Rb +
from a source cell (A) to a sink cell (B) across a node (N).
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of cells of Egeria leaf (Erwee and Goodwin 1983). Electron microscopic observations have revealed various
classes of plasmodesmata. There are open or plugged
channels, with or without a desmotubule inside; they can
be with or without neck constrictions on either side; and
sometimes they have sphincter-like structures (Gunning
and Robards 1976, Willison 1976, Evert et al. 1977, Olesen
1979, Overall et al. 1982, Hepler 1982, Kwiatkowska and
Maszewski 1985, 1986). The large variety in the structure
of plasmodesmata may be related to differences in their
functions in intercellular transport.
Some quantitative studies have been performed using
charophyte as plant material (Tyree et al. 1974, Bostrom
and Walker 1975, 1976). Bostrom and Walker found the
intercellular movement of Cl~ in Chara had a rate of transport of 4 to 60pmols~', with the actual rate depending
markedly on the rate of cytoplasmic streaming.
A major unsolved problem in intercellular transport
concerns the way in which plasmodesmata regulate intercellular transport. Intercellular movement of fluorescent probes has been demonstrated to be inhibited by Ca 2+ ,
inositol triphosphate and the tumor promoter 12-Otetradecanoyl acetate (Erwee and Goodwin 1983, Tucker
and Rosenbaum 1987, Baron et al. 1988). Zawadzki and
Fensom (1986b) found that an intercellular pressure gradient between two tandem internodal cells of Nitella inhibited the transnodal transport of l4C-labeled compounds. Furthermore, Cote et al. (1986) found that nodal
electrical resistance increased when a pressure gradient was
applied across nodes of Chara. From these results, they
proposed that an active mechanism, perhaps an actinmyosin system, is involved in intercellular transport.
Here we present results of our studies of the transport
of R b + , which are based on a newly developed lowtemperature loading method. These results together with
our electrical measurements define the contribution of cytoplasmic streaming and plasmodesmata to the transnodal
transport of Rb + in Chara. Rubidium can be used as a
tracer of potassium, which is the most common cation in
plant cells. The mechanism of intercellular transport of
K + is assumed to be different from that of carbohydrates
because of their different sizes and physiological functions.
Transnodal conductance of Chara corallina
Sakano and Tazawa 1984).
The percentage of Rb + transport from the source to
the sink was calcu-lated from the following equation
(Zawadzki and Fensom 1986a):
Rb transport--.
R
!!
x 100
(1)
Where Rb^ and Rbsi are the total amounts (in mole) of Rb
in the source cell and the sink cell, respectively (note that a
value of 50% is the maximum that can be achieved as a
result of passive diffusion alone).
Extraction and measurement of levels of ATP—Cells
were treated with CB or exposed to low temperature for
3 hours and then frozen in liquid nitrogen. Then they were
boiled in 2 ml of an extraction buffer for 5 min. The extraction buffer contained 25 mM HEPES, 10 mM EDTA, and
0.3% H2O2 (pH was adjusted to 7.4). ATP was analyzed by
the firefly-flash method with an ATP photometer
(Chemglow photometer J4-7441, Aminco, Silver Spring,
MD, U.S.A.).
Measurements of the electrical resistance across the
node and the membrane potential and membrane resistance of internodal cells—The measuring system is shown in
Figures 2a, 2b. Tandem cells were set up in a three-compartment chamber. The three compartments A, N, B were
separated with silicone grease applied in the two grooves of
0.5 cm. Compartments A and B were filled with APW. N
was filled with paraffin oil to reduce the leakage of current
which might cause an under-estimation of the nodal resistance. The membrane potentials EA and EB were measured
with two pairs of electrodes. The tips of glass
microcapillary electrodes were inserted into the vacuoles of
B
Fig.
2b
Fig. 2a Schematic diagram of the experimental system for measuring the nodal electrical resistance, and the equivalent electrical circuit of the nodal region.
Glass microcapillary electrodes fiA and /JB were inserted into internodes A and B, respectively. The membrane potentials EA and EB, and the potential difference across the node, EN, were measured and recorded by a multichannels pen
recorder. Current pulses (I) were applied between the two chambers A and B. rN and r, indicate the specific resistance of the node and
cell sap, and their sum RN is the transnodal resistance.
Fig. 2b Experimental system for measuring the intercellular resistance i^, between node N and internode A.
Two electrodes //A
and nB were first inserted into internodes A and B, respectively, at positions near the node, then one of them, fiB, was pushed forward into the nodal cell.
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with an eyepiece micrometer throughout each transport experiment.
In order to apply a gradient of turgor pressure across
the node after Rb + loading, the chamber that contained
sink or source cell was filled with a solution of 50, 100, 180
or 240 mM sorbitol in APW. In other experiments, both internodal cells were treated with 180 and 240 mM sorbitol, to
test whether the effect of sorbitol was a result of the
pressure gradient between two internodes or was a consequence of a change in turgor in the internodes. To prevent
transcellular movement of water, the nodal compartment
was exposed to moist air, as was also the case in control experiments. The cellular osmotic pressure was measured
with a vapor-pressure osmometer (5100CXR, Wescor Inc.,
UT, U.S.A.).
The Rb + in the cytoplasm and vacuoles of internodal
cells was separated by the vacuolar perfusion technique
(Tazawa et al. 1974). A tandem pair of internodes separated one from the other was made by ligating the internode with polyester thread at loci near the node. Each internode was separated from the node by cutting. Both
ends of an isolated single internode were amputated and
the cell sap was collected with a glass microcapillary tube
(Sakano and Tazawa 1984). Then the vacuole was perfused three times with 20 fi\ of a medium that contained
300 mM sorbitol and 10 mM CaCl2. The remaining cytoplasm was added with deionized water to extract the ions.
Rb + in the cell sap and cytoplasmic fractions was analyzed
by atomic absorption spectrophotometry (PERKINELMER spectrophotometer 370). In order to calculate the
various concentrations, the cytoplasm was assumed to occupy 5% of the total cell volume (Mimura et al. 1984,
741
D. Q. Ding and M. Tazawa
742
the internodal cells A and B, and the reference electrodes
filled with 100 mM KCl-agar were placed in APW. EN is the
nodal potential. Current pulses (I) of 5 x 10~8 A were passed from chamber A to B through the cells via a pair of AgAgCl wires, and the resulting voltage changes eA, eB and eN
were used to calculate the specific membrane resistances rA
and rB, and the sum of the nodal and sap resistance R (R
equals the transnodal electrical resistance) from the following equations:
(R m ) A =e A -a A /I
(R m ) B =e B a B /I
(2)
where aA and aB are the surface areas of internodal cells A
and B, aN is the nodal cross-sectional area calculated from
the average diameter of cells A and B. The two glass electrodes inserted into the cells were separated from each
other by 2.6 cm. In order to measure the resistance of sap,
a long, single internodal cell was set into the chamber in the
same way as the tandem cells. The resistance of sap was
calculated from the following equation:
s=
esas/I
(3)
Then, the nodal resistance rN could be calculated as
follows:
= R-rs
(4)
Upon the application of a gradient of turgor pressure
across the node, R and rN increased to R' and rN', respectively. The relative conductance of a node under a
pressure gradient can then be expressed by equation (5).
(5)
where gN and gN' are the values of electrical conductance of
the node before and after the gradient of turgor pressure
was applied, respectively.
The electrical resistance between the node and internode was also measured by inserting one of the two electrodes into the nodal cell (Fig. 2b). We first inserted the
two electrodes nK and ftB into the tandem internodes A and
B, respectively, at a position very close to the node N.
After recording the nodal resistance rN, the electrode in the
cell B (JJB) was pushed forward at an angle that pointed to
the center of the node until the resistance decreased suddenly to about half of rN. This change indicated that the electrode had been injected into one of the central nodal cells.
Because there are a lot of plasmodesmata between the
nodal cells (Bostrom and Walker 1975), we assume these
nodal cells to be a single, symplastic cell. So the resistance
recorded at this time represented the resistance between cell
A and node N (rAN). If//B was pushed further more, the resistance changed again to almost zero, indicating that /i A
Results
Transnodal transport of Rb+—After treatment of the
source cell with 100 mM RbCl for one hour at 5°C, no Rb +
could be detected in the sink cell. Therefore Rb + could be
loaded at a high concentration into the source cell, with the
level of Rb + in the sink cell remaining at almost zero. As
the incubation temperature was increased from 5°C to
25°C, the transnodal transport of Rb + began (Fig. 3).
After 30min of loading, Rb + had accumulated in the
source cell at a concentration of 43 ± 9 mM in the cytoplasm
and 2.5 ±0.5 mM in the vacuole (Fig. 4). In the first hour,
the transport of Rb + from the source to the sink cell occurred rapidly and then it slowed down gradually (Fig. 3, 4).
After 1 hour of transport, about 17% (Fig. 3) of Rb + loaded in the source cell was transported into the sink cell. At
this time, the concentration of Rb + in the cytoplasm of the
sink cell has increased from 0 to about 3.3 mM and then
remained constant (Fig. 4). Within this period the concentration of Rb + in the vacuole of the sink cell increased constantly. In the source cell, the cytoplasmic level of Rb +
decreased sharply from 43 mM to 11 mM in the first hour
and then slowly in the following two hours (Fig. 4). The
concentration of Rb + in the vacuole of the source cell increased in the first hour, as a result of the influx of Rb +
from the cytoplasm (Fig. 4), then decreased continuously.
Rb + was not found in the incubation solutions of either
chamber A or B after the transport experiment. Furthermore, the total amount of Rb + in the cells after 3 hours
was equal to the amount loaded initially in the source cell.
Thus, no Rb + leaked out of the cells during the experimental transport period.
Effects of cytoplasmic streaming on transnodal trans-
Time (hr)
Fig. 3 Time course of transnodal transport of Rb + {%) from
source to sink cell.
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R=eNaN/I
and fiB were in the same cell, A.
To study the effect of cytochalasin E on R, whole cells
in tandem, including nodal cells, were treated with the
agent at 10//g/ml for 20min.
Transnodal conductance of Chara corallina
743
cr
2
20
40
60
80
Streaming rate (^.m/sec)
3
Time (hr)
Fig. 4 Changes in Rb + concentrations in cytoplasm (square)
and vacuole (circle) of source (close symbols) and sink (open symbols) cells.
port of Rb+—The rate of cytoplasmic streaming can be
varied by changing the temperature and by treating cells
with different concentrations of cytochalasin B. A good
correlation was found between the streaming rate and
temperature (Fig. 5). In this experiment, the temperature
of both tandem cells was changed at the same time. No
significant difference in the streaming rate was found between source and sink cells. The transnodal transport of
Rb + was inhibited by decreases in temperature (Fig. 6).
As shown in Figure 7, the rate of cytoplasmic streaming was maintained at various steady values for 3 hours,
corresponding to different concentrations of CB. The low
rate of streaming in the first lOmin was a consequence of
the low temperature applied during the loading period.
Figure 8 demonstrates that the transnodal transport of
Rb + is greatly influenced by the streaming rate in both the
source and the sink cells. Transnodal transport of Rb +
5
10
15
20
100
Fig. 6 Transnodal transport of Rb + in tandem internodes in relation to rates of cytoplasmic streaming.
To change the streaming rate, whole tandem cells were exposed to various temperatures
for 1 (square) or 3 h. (circle).
seemed to be inhibited by CB to the same extent, irrespective of whether CB was applied to the source cell or the sink
cell. When the streaming was inhibited by the presence of
CB in both the source and sink cells simultaneously, the
transnodal transport of Rb + was reduced more strongly
than in the cases where only the streaming in one internode
was inhibited (Fig. 8). When the streaming rates in source
cells were greater than 70^m/s, the amount of Rb + transport reached a constant value of about 30-40% of the loaded Rb + . In the one-hour experiment, every treatment gave
a similar pattern of transport irrespective of whether CB
was added to one cell or to both cells (Fig. 8, open symbols). When the cytoplasmic streaming in both source and
sink cells was modified simultaneously, either by subjecting
cell to low temperature or by applying CB, the inhibition of
the transport of Rb + correlated with the streaming rate in a
similar fashion, irrespective of the method used.
25
Temperature (°C)
Fig. 5 Changes in rate of cytoplasmic streaming at different
temperatures.
Time (hr)
Fig. 7 Changes in rate of cytoplasmic streaming after treatment
with various concentrations of cytochalasin B.
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I
D. Q. Ding and M. Tazawa
744
100
Fig. 8 Changes of the transnodal transport of Rb + at different
streaming rates.
(O " ) : Only source cells were treated with
CB. (A A): Only sink cells were treated with CB. (O • ) : Both
source and sink cells were treated with CB. The transport was
allowed to proceed for 1 (open symbols) or 3 hours (close symbols).
The effects of low temperature and cytochalasin B on
levels of ATP—The levels of ATP were measured in cells
after treatment with CB or with low temperatures (Table
1). There was no significant difference in the levels of ATP
in cells exposed to different temperatures in the range of
0°C-25°C. Also no decrease in the level of ATP was
found when cells were treated with 10-30/ig/ml CB; a
small increase was observed in cells treated with higher concentrations of CB. Thus, the inhibition of transnodal
transport of Rb + at lower temperatures or by treatment
with CB is not caused by a decrease in the levels of ATP
which might be involved in the closing of plasmodesmata.
The effect of a gradient of turgor pressure across the
node on transnodal transport of Rb+—The osmotic
pressure of internodal cells was found to be 243 ± 10 (n =
30) mOsm. When the turgor pressure was lowered by ap-
Table 1 Levels of ATP in internodal cells of Chara under
different condition
T°C
CB
0
10
20
30
0
10
20
25
ATP (mu)
0.85 ±0.06
0.88±0.09
0.98±0.03
0.99 ±0.09
0.82±0.05
0.82 ±0.06
0.92±0.05
0.85 ±0.07
(5)
(5)
(5)
(5)
(5)
(5)
(5)
(5)
50
100
150
200
Turgor pressure gradient (mOsm)
250
Fig. 9 Transnodal transport of Rb + over the course of one hour
in relation to the gradient of turgor pressure imposed between two
tandem internodes.
(•): Both tandem internodes were treated
with 180 mil sorbitol. (A): Only the sink cell was treated with sorbitol. (D): Only the source cell was treated with sorbitol.
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20
40
60
80
Streaming rate (^.m/sec)
plication of 180mM or even as much as 240 mil sorbitol to
both of the tandem internodes, the transport of Rb + was
not affected (Fig. 9). However, when a gradient of turgor
pressure was set up between the two internodes, the
transnodal transport of Rb + was inhibited to a significantly
extent (Fig. 9), regardless of whether it was the source or
the sink cell that was treated with sorbitol. When 240 mM
sorbitol was applied to the sink cell, the cell almost completely lost its turgor pressure, and the transnodal transport of Rb + was reduced to only 3% of control values.
However, the rates of cytoplasmic streaming of the sink
cells were only reduced by about 10%. Therefore, it is
neither the sorbitol itself nor the decrease in turgor
pressure and the decrease in streaming rate that influence
the transport, but it appears to be the intercellular gradient
of turgor pressure between the source cell and the sink cell
that inhibits transport of Rb + .
Electrical resistance—The transnodal electrical resistance can be measured when two glass needle electrodes are
inserted into the vacuoles of the two tandem internodal
cells (Fig. 2a). Internodal cells of Chara had a membrane
potential of -158 ± 18 mV (n = 12), and a membrane resistance (rA, rB) of 5,7O0± 1,400 mJ2m 2 (n= 15) when immersed in APW. The specific transnodal resistance, R, was
44 ± 8 mJ2m 2 (n= 15). R includes both the nodal resistance rN and the sap resistance rs. The sap resistance rs was
found to be 18±1 mQm2 (n = 5). Therefore, the resistance of the node rN was calculated to be 26 mQ • m2. When
the nodal resistance was measured as shown in Figure 2b,
we obtained a value of 21 ±6.5 mQm1 (n = 6) (Table 2).
The relative transnodal conductance (gN7gN) decreased as the gradient of turgor pressure across the node increased (Fig. 10). When the pressure gradient was
180 mOsm, the electrical conductance was only about 50%
Transnodal conductance of Chara corallina
Table 2 Transnodal resistance rN and resistance of the
junction side rAN between the internode and node
Resistance
Number
of cells
rN
A: APW
B: APW
21.3 + 6.5 mQm2
6
rAN
A: APW
B: APW
8.4±2.2mflm 2
8
rAN
A: 200 mM sorbitol
B: APW
27.1±9.6mfim 2
7
rAN
A: APW
B: 200 mM sorbitol
8.2±2.8 mi2-m2
5
r^
A: 200 mM sorbitol
B: 200 mM sorbitol
10.2±1.9mJ2m 2
4
For explanations of A, B and N, refer to Fig. 2B.
TAN-
of that of the control. We did not detect a further
decrease even when internodal cells lost further turgor in
250 mM sorbitol. The increase in nodal resistance could be
reversed by removal of the pressure gradient. However,
when the process of applying and removing the pressure
gradient was repeated several times, the cells became insensitive to the pressure gradient. Both the increase in and the
recovery of the resistance happened within 2-5 min after
changing the solution, but it took 20-40 min or more to attain a constant value. The electrical potential difference between the two microcapillary electrodes (EN), measured
under zero current, changed by about 10 mV upon imposition of a gradient of turgor pressure. This change resulted
mostly from changes in the membrane potential of the cell
to which the osmotic stress was applied. The membrane
50
100
150
potential and membrane resistance of the internodal cells
did not change significantly when the cells were under
osmotic stress. Thus, it is the increase in resistance of plasmodesmata that causes the decrease in transnodal conductance.
In order to determine the site responsible for the
change in resistance, the intercellular resistance of the junction side (t/w) between the internode and the node was
measured by inserting one electrode into the center nodal
cell (Fig. 2b). As shown in Table 2, rAN is about half of the
transnodal resistance rN in APW, and rAN did not change
markedly when both sides of the internode were exposed to
200 mM sorbitol. When one side of the tandem internodes
was treated with sorbitol, only the resistance of the junction on this side increased, while the other remained unchanged. Therefore, the increase in rN induced by the gradient of turgor pressure is actually the result of increase in
200
250
Turgor pressure gradient (mOsm)
Fig. 10 Dependence of transnodal electrical conductance {%)
on the gradient of turgor pressure across the node caused by concentration of 50 to 240 mM sorbitol.
Each value is the average
of results from 3 to 6 cells, with the standard deviation.
Cytochalasin E, which inhibits cytoplasmic streaming
more effectively than CB (Wayne and Tazawa 1988),
affected neither the membrane resistance nor the nodal resistance. After the cessation of cytoplasmic streaming in
both the internodal and nodal cells, a gradient of turgor
pressure of 180mOsm was applied. The nodal resistance
increased to the same extent regardless of whether cells
were treated with CE or not. Thus, cytoplasmic streaming
does not influence the electrical resistance across the node.
It can, therefore, be deduced that the plasmodesmata remain open even when cytoplasmic streaming has stopped.
CE did not have any direct effect on the plasmodesmatal activity. The inhibition of transnodal transport of Rb + by
CB appears, thus, to be a result of the inhibition of cytoplasmic streaming.
Discussion
Apparent permeability to Rb+ and diffusion coefficient of the node—The new method of low-temperature
loading allowed us to separate the loading process from the
transport of R b + . This separation enables us to estimate
the transnodal permeability or the diffusion coefficient of
Rb + across the node, in terms of the gradient of concentrations of solutes between the cytoplasm of the source and
sink cells, if we assume that the transnodal transport of
Rb + is a passive process. The determination of the concentration of Rb + in the cytoplasm, without contamination by
the cell sap, was achieved in the present study by isolating
the cytoplasm from the cell sap by the vacuolar perfusion
method. Contamination by the cell sap causes errors in
the estimation of the rate of transnodal transport, as suggested by Bostrom and Walker (1975).
Forty minutes after initiation of transport, we measured the transnodal flux of Rb + (JRb) to be 1.2 x 10"5 molm~ 2 -s~' if the nodal cross-sectional area is estimated from
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Treatment
745
746
D. Q. Ding and M. Tazawa
the diameter of internodal cells. JRb is assumed to be
dependent on the gradient of concentration of Rb + and its
apparent permeability (P Rb ). Then
(6)
(7)
Where L is the transnodal length of plasmodesmata and is
assumed to be equal to the sum of the thickness of the
transnodal cell walls. From the data of Bostrom and
Walker (1975) and Fischer et al. (1974), L is taken to be
Then, D Rb in equation (7) is calculated to be 2.3 x
10~12 m2 -s" 1 . Since the fraction of the area of the cell wall
occupied by plasmodesmata is between 5% (Bostrom and
Walker 1975) and 15% (Fischer et al. 1974) in nodal cells of
Chara corallina nodal cells, we chose 10% as an average
value. Using this value, we calculated the diffusion coefficient of plasmodesmata per se to be about 2.3 x 10"" m2s~", which is equivalent to 1% of the diffusion coefficient
of RbCl in water (2x 10~ 9 m 2 s~') (Robinson and Stokes
1959). The very low diffusion coefficient of Rb + through
plasmodesmata is compatible with the assumption that the
transport of Rb + through plasmodesmata is a passive diffusion process.
The role of cytoplasmic streaming in transnodal transport o/Rb+—The present study clearly demonstrated that
cytoplasmic streaming is indispensable for the transnodal
transport of Rb + in Chara corallina. The streaming in
both the source and sink cells contributed to the transport
by establishing and maintaining a concentration gradient
between two internodal cells. In the source cell the streaming supplies R b + quickly to the nodal junction, while in the
sink cell the streaming sweeps away Rb + that reached it
from the source cell via the nodal junction. In the case of
the 3 h experiment whose results are shown in Figure 8,
treatment with CB of both source and sink cells reduced
the intercellular transport of R b + more than treatment of
just source or sink cell. However, in the case of the 1 h experiment, no difference was observed between results of
treatment of both cells or of one cell with CB. In the case
of the 1 h experiment, the gradient of Rb + between source
and sink cell was larger than that in the case of the longer
experiment. This difference may be responsible for the
Downloaded from http://pcp.oxfordjournals.org/ at Penn State University (Paterno Lib) on September 12, 2016
where C^ and C^, are the concentrations of Rb + in the cytoplasm of the sink and source cell, respectively. During the
first 40 min of transnodal transport, Csi increased from 0 to
2.8 mM, while C^ decreased from 43 mM to 13 rrm. Adopting the mean concentrations of C^ and Cso of 1.4 mM and
28 mM, respectively, using equation (6) we get a value of
4.6 x 1 0 ~ 7 m s ~ ' for P Rb . The apparent diffusion coefficient of Rb + across the node (DRb) can be calculated from
the following equation:
difference in the responses of the transport of Rb + to treatment with CB between the 1 and the 3 h experiment.
Both cytochalasin B and low temperature inhibit cytoplasmic streaming but they do so in different ways. Higher
concentrations of CB rapidly inhibit streaming by
abolishing the motive force (Kuroda and Kamiya 1981).
By contrast, low temperature decreases the streaming rate
by increasing the cytoplasmic viscosity (Tazawa 1968).
Even if the cytoplasmic streaming is inhibited by different
mechanisms, the inhibition of transport of Rb + seems only
to correlate with the rate of cytoplasmic streaming. Thus,
CB or low temperature inhibits the transnodal transport
not directly but indirectly via inhibition of cytoplasmic
streaming. Since CB or low temperature did not
significantly affect the level of ATP, the possibility a
decreased supply of energy for transport through the plasmodesmata can be eliminated. Furthermore, the absence
of any inhibition of transnodal electrical conductance by
CE excludes an involvement of actin filaments in the regulation of plasmodesmatal opening, although movement of
water across the plasmalemma is regulated by the actin cytoskeleton (Wayne and Tazawa 1988). Thus, evidence supporting the notion of active plasmodesmatal transport
(Zawadzki and Fensom 1986b) was not found in the
present work. Cessation of streaming in either node cells
or internodes did not affect the electrical conductance of
the node. Thus, regulation of plasmodesmatal opening is
not related to the rate of cytoplasmic streaming.
Transnodal transport ofRb+ and electrical resistance
in relation to a gradient of turgor pressure—Transnodal resistance (rN) in Chara corallina was found to be 21 to 26
mQ • m2. This value is similar to the values obtained so far
in Chara (15mf2m 2 , Sibaoka 1966; 47mflm 2 , Bostrom
and Walker 1975; and 63 mQm2, Cote et al. 1986), but it is
much smaller than the values measured in Nitella which
ranged from 120mO-m 2 to 170mJ2m 2 (Lou 1955;
Spans wick and Costerton 1967).
In present study we found that the transnodal gradient
of turgor pressure decreases both the transnodal movement
of Rb + and the electrical conductance between the node
and the internode with a lower turgor pressure. Zawadzki
and Fensom (1986b) already reported that the transnodal
transport of 14C-labeled photosynthetic products in Nitella
flexilis was inhibited by a transnodal pressure gradient.
However, this transport seems to be more sensitive to the
pressure gradient than that of Rb + , perhaps because of the
larger molecular sizes of photosynthetic products. Cote et
al. (1986) found that the transnodal pressure gradient imposed by 100mM mannitol increased the transnodal resistance by 30% (i.e. a decrease in the electrical conductance
of 25%) and they assumed a valve-like mechanism to be
operative in plasmodesmata, as originally proposed by
Zawadzki and Fensom (1986b). Using sorbitol, we confirmed their observations, and further showed that (1) the
Transnodal conductance of Chara corallina
We are grateful to Dr. T. Shimmen (University of Tokyo) for
his helpful suggestions during this study. Our thanks are also due
to Dr. R. Wayne (Cornell University) for his critical reading of
and his help with the English text. This work was supported by
Grants-in-Aid for Special Project Research (Nos. 61129001,
62119002) from the Ministry of Education, Science and Culture,
Japan.
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decrease in transnodal electrical conductance is proportional to the magnitude of the gradient of turgor pressure;
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141
748
D. Q. Ding and M. Tazawa
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(Received February 15, 1989; Accepted April 20, 1989)
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