y. Cell Set. 62, 385-405 (1983)
385
Printed in Great Britain © The Company of Biologists Limited 1983
REGULATION OF CYTOPLASMIC STREAMING IN
VALLISNERIA MESOPHYLL CELLS
SHINGO TAKAGI* AND REIKO NAGAI't
Department of Biology, Faculty of Science, Osaka University, Toyonaka, Osaka 560,
Japan
SUMMARY
Induction and cessation of the rotational cytoplasmic streaming in Vallisneria mesophyll cells
could be controlled by external stimuli. In cells that had been kept in darkness the cytoplasm
remained quiescent. However, when the cells were treated in the dark with EGTA solution
(10mM or 20mM buffered with 10mM-Tris-maleate at pH7-0), rotational cytoplasmic streaming
was induced. When the cells were transferred again to artificial pond water in the dark, the induced
streaming was inhibited; that is, only 50 % of the observed cells exhibited active streaming after 2 h.
When the cells were irradiated continuously with far-red light (Am^K = 750nm, 0'4W/m 2 ) in the
same external medium, the induced streaming was inhibited almost completely within 2h.
The relative quantum effectiveness of monochromatic light (450—800 nm) in producing cessation
of streaming was also investigated. Irradiation with light of 450, 550 and 600 nm was almost as
effective as darkness. Light of 500 and 650 nm was less effective than dark exposure. Only irradiation
at 750 nm stopped streaming in almost all cells. But when calcium was excluded from the external
medium, the effect of far-red light decreased to almost the dark control level. Light of 800 nm also
inhibited the streaming but the effect was much less than that of far-red light.
Microfilaments in bundles with the long axis parallel to the streaming direction were localized in
the vicinity of the cell membrane. Their configuration, localization and distribution were the same
in the present experimental system irrespective of whether the cytoplasm was streaming or quiescent.
Intracellular calcium was examined by electron microscopic cytochemistry and X-ray
microanalysis. In cells with streaming induced by EGTA, only a small amount of calciumcontaining precipitates formed in the cytoplasm in the presence of antimony. A few precipitates were
found in the chloroplasts, the middle lamella of the cell wall and at the border between the cytoplasm
and the cell wall. On the other hand, in cells treated with EGTA and subsequently irradiated with
far-red light in artificial pond water, many precipitates were observed in the cytoplasm,
chloroplasts, mitochondria and endoplasmic reticulum. The middle lamella was also heavily
stained.
On the basis of these observations, it was concluded that rotational cytoplasmic streaming in
Vallisneria cells can be induced when the free calcium concentration in the cytoplasm decreases and
that the induced streaming is arrested when the free calcium concentration in the cytoplasm increases. Far-red light accelerates the increase of calcium in the cytoplasm.
INTRODUCTION
In leaf cells of the higher aquatic plants, Elodea and Vallisneria, rotational streaming of the cytoplasm is induced by irradiation with visible light (photodinesis) or
application of various chemicals (chemodinesis). Thus, this type of streaming is called
•Present address: Department of Biology, General Education, Osaka University, Toyonaka,
Osaka 560, Japan.
f Author for correspondence.
386
5. Takagi and R. Nagai
secondary streaming, while the type seen in characean cells is called primary streaming, because it persists under normal conditions (Hauptfleisch, 1892; Kamiya, 1959).
Seitz (1967, 1972) investigated the nature of the photoreceptor involved in the lightinduced movements in Vallisneria and the effect of light on centrifugability of
chloroplasts, cyclosis and phototactic orientational movement of chloroplasts. He
proposed that the primary effect of light is due to regulation of the availability of ATP
from oxidative and photosynthetic phosphorylation; an increased availability of ATP
activating cytoplasmic streaming and thus inducing chloroplast movement.
The microfilament organization responsible for the generation of motive force has
been reported for the epidermal cells of Vallisneria (Yamaguchi & Nagai, 1981).
However, the mechanism by which the induction and cessation of streaming is
regulated remains obscure.
Basically, the mechanics of cytoplasmic streaming in characean internodal cells
seem to be as follows. Rotational cytoplasmic streaming is caused by unidirectional
sliding of endoplasmic organelles along the bundles of microfilaments that are anchored on the stationary chloroplast files (Kamitsubo, 1966; Kamiya & Kuroda,
1956; Nagai & Rebhun, 1966; Nagai & Hayama, 19796). The microfilaments are
mainly composed of F-actin (Palevitz, Ash & Hepler, 1974; Williamson, 1974;
Palevitz & Hepler, 1975). The sliding organelles are equipped with myosin-like
protein (Nagai & Hayama, 1979a,6). Motility requires Mg2+-ATP and Ca2+ at
10~7M or less (Williamson, 1975; Williamson & Ashley, 1982; Hayama, Shimmen &
Tazawa, 1979; Hayama & Tazawa, 1980; Tominaga & Tazawa, 1981).
In the case of Vallisneria: (1) the microfilament bundles are found at the ectoplasmic gel layer of the epidermal cells (Yamaguchi & Nagai, 1981); (2) cytochalasin B
inhibits streaming in the mesophyll cells (Ishigami & Nagai, 1980); and (3) a myosinlike protein has been extracted and partially purified from Egeiia (Elodea) densa
(Ohsuka& Inoue, 1979). These facts are shared by characean cells exhibiting primary
streaming.
This paper aims to show that induction and cessation of cytoplasmic streaming in
Vallisneria mesophyll cells are controlled by a change in Ca2+ concentration in the
cytoplasm and not by a change in microfilament organization.
MATERIALS
AND
METHODS
Plant
Vallisneria gigantea Graebn was purchased at a tropical fish store and cultured in a bucket with
soil at the bottom and filled with tap water. The culture was kept under a 12 h light (4000 lux with
fluorescent Iamps)/I2h dark regime at room temperature.
Pretreatment of samples
To exclude possible induction of streaming by cutting and touching the plants, the following
conditioning was done before the experiments.
A leaf segment, about 10 cm long, was cut from a healthy plant in the stock culture at the end of
the light period. It was then cut into smaller pieces about 2 mm long, which were then floated on
artificial pond water (APW) containing 5 X 10~ 5 M-KC1, 2 X 10~*M-NaCl, 10" 4 M-Ca(NO 3 ) 2 ,
Cytoplasmic streaming in Vallisneria
387
Table 1. Combination of filters used to obtain monochromatic lights
Interference
filter
Wavelength
(nm)
Half-bandwidth (nm)
Cut-off
filter
KL-45
KL-50
KL-55
KL-60
KL-65
KL-70
KL-75
KL-80
451-8
497-5
551-0
595-0
650-0
696-0
747-0
795-0
9-6
13-5
90
14-0
17-5
16-5
18-0
17-0
V-Y43
V-Y48
V-053
V-058
V-R64
V-R68
V-R68
V-R68
10"4M-Mg(NO3)2 and 2 x 10 5M-Tris-maleate buffer at pH7-0. The air trapped in the intercellular space was evacuated, and each piece of leaf was placed in a separate glass vessel with 40 ml
of APW and incubated under the original light conditions of the 12h dark and 12h light regime.
During incubation, the medium was replaced once at the end of a dark period. After one cycle of
dark and light, each specimen was mounted on a glass slide with a coverslip using a small amount
of vaseline at each corner. The air gap between the glass slide and the coverslip was filled with fresh
medium. The slide loaded with the specimen and the coverslip was then immersed in a glass vessel
filled with the same medium and kept in the dark for another 12^18h at 20-25 °C. After these
procedures, the specimens could be used for the experiments without being touched with forceps.
The cytoplasm in all cells was stationary at the end of these procedures.
Light sources
Green light (Amu = 550 nm, 0-6 W/m 2 ) was used as safe light throughout the experiments,
because green light has no effect on induction and cessation of streaming. The green light was
obtained by combining an interference filter (KL-55, Toshiba, Japan) and a cut-off filter (V-053,
Toshiba, Japan). These filters were placed in front of a tungsten lamp. To exclude any heat effect,
a heat filter and a glass tank (17 cm X 9 cm X 9 cm) filled with tap water were placed in the light path.
Other monochromatic light was prepared similarly with the combination of an interference filter and
a cut-off filter (Table 1). The intensity of the monochromatic light was measured with a photodiode
detector (PIN-10DF, United Detector Technology), which was positioned in the focal plane of the
light microscope. The output from the detector was monitored with a voltmeter (SP-H6V, Riken
Denshi, Japan) and later converted into a quantum number. This calculation was performed with
the assumption that all of the energy transmitted by each interference filter corresponded to its
wavelength maximum.
Irradiation with monochromatic light
The specimen was irradiated through a condenser lens on the microscope stage with each
monochromatic light at a constant quantum number of 1 -53—1 -58(X 10 l8 )photon/s.m 2 under a
given set of test conditions. When the specimen was irradiated with light of over 700 nm, observations were made using a TV camera (CTC-5600JC, equipped with Newvicon, Ikegami, Japan) and
a monitor TV (WV-930, National, Japan).
Application of chemicals
Ethvlencglycol-bis(2-aminoethylether)-./V,A',A'',A''-tetraaceticacid (EGTA) (Wako, Japan) was
dissolved in distilled water at 200 mM with potassium hydroxide to adjust the pH to 7-0, then diluted
with lOniM-Tris-maleate buffer (pH7-0) to give final concentrations of 1, 10 and 20 mM. EGTA
was applied under dark conditions by gentle irrigation between the coverslip and the slide. The
induction of streaming was examined under green light.
388
5. Takagi and R. Nagai
Measurement of effectiveness
Specimens were examined with a Nikon light microscope equipped with 40 X objective and SX
ocular lenses. The effectiveness of irradiation and chemicals on induction or cessation of streaming
was expressed as the ratio of streaming cells to the total of observed cells. Fifty to a hundred cells
from four to ten different leaf segments were examined for each test condition. Cells were counted
as streaming when their chloroplasts exhibited streaming continuously for at least 5 s.
Electron microscopy
The entire specimen was illuminated and induction or cessation of streaming in all cells were
examined before the specimens were transferred to the fixative.
The specimens were prefixed with 2-5 % glutaraldehyde buffered with 25 mM-cacodylate
(pH 7-0) for 2 h, under evacuation for 10 min at the beginning of the fixation. After washing twice
with the buffer, the specimens were post-fixed for 2h with 2 % OsO* in the same buffer, under
evacuation as before. The irradiated specimens were prefixed in far-red light (Am*, = 750 nm, 0-4 W/
m 2 ), then kept in the dark. EGTA-treated specimens were fixed in the dark or under green light.
To examine the intracellular localization of calcium, the specimens were fixed in the same way
as above, but 2 % potassium pyroantimonate (Wako, Japan) was added throughout prefixation,
postfixation and washing, and the concentration of the buffer solution was reduced to 20 itiM.
After dehydration through a graded series of ethanol, all specimens were embedded in Spurr's
(1969) medium, then sectioned. Thin sections were stained with uranyl acetate and lead citrate. For
the examination for calcium precipitates, the sections were not stained. A JEM 100-C type electron
microscope was used for observations at 80 kV. For X-ray microanalysis, a Hitachi X-600 type
electron microscope and KEVEX 7000Q energy analyser were used.
RESULTS
Induction of streaming
A Vallisneria leaf is made up of epidermal cells on each surface and the inner part
is occupied by mesophyll cells. A cross-section shows that the middle area of the leaf
consists of several layers of mesophyll cells (Fig. 1A) and there are two layers near the
leaf edges (Fig. 1B). In all experiments observations were made on mesophyll cells
near the leaf edges. Cytoplasmic streaming could be induced more easily in mesophyll
cells than in epidermal cells and it was desired to keep disturbance of actinic light at
a minimum.
In mesophyll cells, no cytoplasmic streaming was observed at the end of the conditioning period in darkness, apart from slight movements of some cytoplasmic particles. Chloroplasts remained still and were distributed almost evenly in the cell. The
nucleus was anchored at an unspecified locus in the cytoplasm.
When treated with EGTA solution, agitational movements of the cytoplasmic
particles immediately became vigorous and some underwent translatory motions. In
time, some chloroplasts began to follow these local streamlets of cytoplasmic particles,
but the movement was haphazard. They moved 10-20/zm at a time and then stopped.
This kind of saltatory movement, as named by Rebhun (1964), was repeated in each
chloroplast for a while. At 1—2 min after EGTA had been applied, the chloroplasts
gradually formed a line and began to move continuously. Then the cytoplasmic
streaming formed a closed circuit, which rotated along the cell wall unidirectionally,
either clockwise or counter-clockwise. The streaming rate was low at the beginning
Cytoplasmic streaming in Vallisneria
389
Fig. 1. Photomicrographs of cross-sections of Vallisneria leaf. A. A middle area where
several layers of mesophyll cells (meso) are found between the two layers of epidermal cells
(epi). B. An area near the leaf edge where only two layers of mesophyll cells are found
between the two layers of epidermal cells, n, nucleus; c, chloroplast; p, cytoplasmic
particle. Bar, 100^m.
but reached a maximum value in the range of 10—20^im/s within about lOmin. The
nucleus could rarely be observed in the quiescent cell because of its position. In
several cases it was observed to be remaining still, but it began to move on application
of EGTA, as with the chloroplasts, and finally participated in the active rotational
cytoplasmic streaming. Once the cytoplasmic streaming was established, the nucleus
390
5. Takagi and R. Nagai
could be observed very clearly moving along the cell wall, together with cytoplasmic
particles and chloroplasts.
To express quantitatively the relationship between EGTA concentration and the
induction of streaming in the dark, the number of cells (iVx , Fig. 2) with cytoplasmic
streaming was counted under green light after appropriate durations of treatment and
the ratio of the streaming cells to the total observed cells (iVtoUi, Fig. 2) was plotted
as percentage against time.
In 1 mM-EGTA, the number of streaming cells increased gradually with time. The
Arx/Artotai ratio reached a plateau at 50% after treatment for 1 h. In 10mM-EGTA,
streaming was observed in all cells within 30 min. This active state was maintained for
2—3 h. With 20 mM-EGTA, streaming was induced more quickly than with 10mM,
but the active state lasted only around 30 min before it started to decline. With the
buffer solution alone, no streaming was induced. Thus, EGTA is clearly responsible
for the induction in dark conditions.
Cessation of streaming produced by irradiation with light of various wavelengths
Since the actinic light for stopping streaming was not known, the effectiveness of
monochromatic light of various wavelengths was investigated.
30
Time (min)
60
Fig. 2. Induction of streaming in the dark with EGTA. The number of streaming cells
(Afx) was counted under green light after various periods of treatment. The ratio of cells
involved in streaming to total cells observed (NM,\) is plotted as the percentage against
time (min). (O
O) 1 mM-EGTA (A'IOui. 58; number of specimens iV,, 4);
(•
• ) 10niM-EGTA (AU.i, 106; N., 8); (A
A) 20mM-EGTA (i\\M.\, 53;
t) lOmM-Tris-maleate buffer (ArtOui, 55; Nt, 4), control.
(Jytoplasmic streaming in Vallisneria
391
First, specimens, in which all cells exhibited streaming in the dark due to treatment
with lOmM-EGTA, were continuously irradiated with far-red (f.r.) light (Amax =
750 nm, 0-4 W/m2) after the EGTA solution had been replaced with APW. As shown
in Fig. 3 (O
O), the number of streaming cells decreased with increase in irradiation time. When the specimens were kept in the dark the number of streaming
cells also decreased, but much more slowly (Fig. 3, #
• ) . Clearly, far-red
light had a definite effect in producing cessation of streaming.
Next, the specimens were treated in the same manner, then were individually
irradiated continuously with monochromatic light of a different wavelength at the
same quantum number to obtain the time-course of the cessation of streaming. The
values of [(Nx/NVM\)iMk
— (Nx/Ntoit\)m<mo] / (Nx/Ntott\)dtTk were plotted as the relative
quantum effectiveness of different wavelengths (Fig. 4). Positive values mean that
streaming was inhibited more by the particular type of light than by the dark treatment, while negative values mean that the streaming was inhibited less. The unbroken
line represents the values obtained after irradiation for 1 h and the broken line those
after irradiation for 2h.
Clearly, the streaming was inhibited most effectively and specifically by far-red
100
60
Time (min)
80
100
120
Fig. 3. Cessation of streaming by irradiation with far-red light. All specimens were treated
with lOmM-EGTA in the dark to induce streaming in all cells (100%). After removing
EGTA, some specimens were irradiated continuously with far-red light in APW
(O
O). Some were kept under dark conditions in the same medium ( •
#).
The streaming cells in each case were counted at intervals of about 20 min and the ratio
A\/A'tm,i is plotted as the percentage against time. The length of the vertical bar is the s.E.
of each value. M««i > 54-76; A',, 7-10 for the irradiation test: A'lOui, 68-109; N,, 5-8 for
the dark test.
392
S. Takagt and R. Nagai
10
0-8
, / \
0-6
<D
C
<D
I
/A \
0-4
0-2
CD
E
c
0
A
jj
CD
D
CJ
CD
j? - 0 - 2
\
<B
-0-4
-0-6 •
\
/ '
it
450
\
\
/
\
\
\
/
500
/
/
550
600
\
\
/
J
V
650
700
750
800
Wavelength (nm)
Fig. 4. Relative quantum effectiveness of monochromatic light on the cessation of streaming. All specimens were treated with EGTA in the dark to induce streaming in all cells.
After removing EGTA, they were separated into nine groups. One group of specimens was
kept under dark conditions to obtain the time-course of the cessation of streaming in APW.
The other groups were individually irradiated continuously with monochromatic light of
a different wavelength (mono) at a constant quantum number to obtain the time-course
of the cessation of streaming. The values of [(Ns/A'toui)ivk— (Nx/NMd)mono] /(N\/Ntoiti)i,*
were obtained and plotted as the relative quantum effectiveness of wavelengths. The
unbroken and broken lines represent values obtained after l h and 2h of irradiation,
respectively. Aftoui, 51-76; N,, 5-10 for each irradiation test: Ar,m.i, 95-109; A',, 7-8 for
the dark test.
light. Light of 450, 550 and 600 nm had no effect — that is, the ratio of streaming cells to
the total observed cells took the same time-course as that observed in the dark. Light of
500 and 650 nm had less effect in causing cessation of streaming than the dark control.
Light of 800 nm inhibited the streaming but the effect was much less than that of far-red
light. The inhibitory effect caused by irradiation with far-red light was enhanced with
increasing time.
Microfilament organization
Microfilaments have been postulated as generating motive force in Vallisneria
mesophyll cells, because streaming is inhibited by cytochalasin B (Ishigami & Nagai,
1980). Ishigami & Nagai observed that the direction of the streaming was reversed in
50% of the total cells observed after long-term treatment with cytochalasin B. They
Cytoplasmic streaming in Vallisneria
393
interpreted this phenomenon as showing that the microfilament bundles had been
completely destroyed during the period when streaming ceased and was re-established
after removal of the drug, because the streaming direction is known to be determined
by the polarity of F-actin (Kersey, Hepler, Palevitz & Wessells, 1976).
This is also supposed to be the case for irradiation with far-red light. To confirm
this, the configuration, localization and distribution of the microfilaments in cells
were examined and compared at the end of each successive treatment: namely, (a)
after the pretreatment in which the specimens had been kept in the dark for 12-18 h;
(b) after they were treated with 10 mM-EGTA; and (c) after they were irradiated with
far-red light.
In cells under condition (a), the cytoplasm did not show any sign of streaming. In
these cells, bundles of microfilaments, which are very similar in appearance to those
in epidermal cells of Vallisneria (Yamaguch & Nagai, 1981), characean internodal
cells (Nagai & Rebhun, 1966; Nagai & Hayama, 19796) and higher plants (Parthasarathy & Miihlethaler, 1972; O'Brien & Thimann, 1966), were localized in the vicinity of the cell membrane and arranged parallel to the direction in which the cytoplasm
is expected to stream. Fig. 5A shows part of two adjacent mesophyll cells in crosssection at low magnification. Fig. 5B is a magnified picture of Fig. 5A. Cross-sections
of the microfilaments (mf) appear as 30-50 closely packed electron-dense dots. The
distance from one bundle to the next varied with the cytoplasmic layer of the cell. This
cannot be attributed to poor fixation, because the cytoplasm seems to be generally well
preserved.
Under condition (b), the cytoplasm was actively streaming in all cells. The morphology of the microfilament bundles, shown in Fig. 5c, was similar to that under
condition (a). Bundles were found at the site where streaming occurred. In the
specimens under condition (c), streaming was completely inhibited. The microfilament bundles also remained unchanged in their appearance, localization and
distribution (Fig. 5D).
From these observations, we conclude that cessation of the streaming in the dark
or cessation produced by irradiation with far-red light cannot be explained in terms
of changes in the microfilament arrangement.
Role of calcium in the control of streaming
The most plausible factor responsible for controlling the streaming is the Ca2+
concentration in the cytoplasm. Many reports have indicated that various kinds of
movements in eukaryotic cells are regulated by Ca2+. Also, red and far-red light affect
Ca2+ movement across the cell membrane (Dreyer& Weisenseel, 1979; Hale&Roux,
1980).
From this point of view, we examined the effect of external Ca2+ on cells irradiated
with far-red light. First, the specimens were treated with EGTA in the dark to induce
streaming in all cells. Some were then transferred to calcium-containing APW and
irradiated continuously with far-red light. Other specimens were irradiated in
calcium-free APW. Fig. 6 shows the time-course of the ratio of streaming cells to total
cells observed in the presence or absence of Ca2+. Clearly, the induced streaming was
394
5. Takagi and R. Nagai
Fig-5
Vyloplasmic streaming in Vallisneria
395
100
\
80
\
^^L^
^
\
VQ
\
1
\
S
N
\
60
1
40
20
)
20
40
60
Time (min)
80
100
120
Fig. 6. Effect of calcium on cessation of streaming. Specimens were treated with EGTA
in the dark to induce streaming in all cells (100%). They were then divided into four
groups according to the experimental conditions: (1) calcium-free APW in the dark
( A - - - A ) ; (2) calcium-containing APW in the dark ( •
• ) ; (3) irradiated with farred light in calcium-free APW (A — A ) ; and (4) irradiated with far-red light in calciumcontaining APW (O
O). The ratio A'x/A/l0U| is plotted as percentage against time of
treatment. The length of the vertical bar is the S.E. of each value. A'mui for condition (1)
and condition (3) was 87-102 and 40-57, respectively, and A', was 6-7 and 5-7. As for
the S.E., A'toU| and A', for condition (2) and condition (4), see Fig. 3 and its legend.
markedly inhibited only by the combined action of Ca2+ and irradiation with far-red
light. Only 10% of the cells continued streaming after 2h irradiation (O
O).
2+
In the absence of Ca , the inhibition was about the same as that in the dark
(A — A). The time-course in the dark was almost the same in the presence or absence
of Ca2+ ( •
• , A---A).
The inhibitory effect of Ca2+ was confirmed further by the following experiments.
All specimens were treated with EGTA as before. The specimens were then transferred to either calcium-containing or calcium-free APW for irradiation with far-red
Fig. 5. Electron micrographs of microfilament bundles (»«/). About 30—50 closely packed
dots, presenting a cross-section of microfilaments, are localized near the cell membrane.
The distance from one bundle to the adjacent one varies, A. Part of two adjacent mesophyll
cells, kept in the dark for 12— 18 h, in which the cytoplasm did not show streaming. The
fixed cytoplasm seems to be well preserved, B. A higher magnification of A; C, microfilament bundles in cells treated with EGTA, in which the cytoplasm was actively streaming;
D, microfilament bundles in cells treated with EGTA and subsequently irradiated with farred light, in which the streaming was completely inhibited. Bars: A, 0-5 fxm\ B-D, 0-1 l-im.
396
5. Takagi and R. Nagai
—o
60
120
180
240
300
Time (min)
Fig. 7. Repetition of induction and cessation of streaming. Each specimen kept in the dark
showed no streaming. Streaming was induced in all cells by treatment with EGTA in the
dark (O
O). After removal of EGTA with calcium-free APW (A) or with calciumcontaining APW (B) , each specimen was irradiated with far-red light (O — O). When the
cells were treated again with EGTA in the dark at the time streaming stopped, they
resumed streaming. These cessation and reactivation cycles could be repeated. Artoui was
5 in A and 6 in B.
light. When the cells were treated with EGTA again at the time the cytoplasm ceased
to flow, they resumed streaming. Inhibition and reactivation of streaming could be
repeated several times.
In the presence of Ca2+ (Fig. 7B), streaming stopped within 80 min on each irradiation. However, in the absence of Ca2+ (Fig. 7A), it took longer, about 160 min, before
streaming came to a complete standstill. Reactivation of streaming by EGTA could
be repeated but streaming did not stop completely on the second irradiation, even
after 3 h.
Thus, streaming stopped more rapidly in the presence of Ca2+ when the cells were
irradiated with far-red light and the effect of the light was completely removed by
Fig. 8. Electron micrographs showing intracellular calcium deposits in cells kept in the
dark for 12-18 h before fixation in the presence of potassium pyroantimonate. Precipitates
are formed in the vacuole, cytoplasm (cyt) and the middle lamella (ml) of the cell wall
(cw). The sections were not stained, A. Cross-section of cells showing the distribution of
precipitates at low magnification, B. Cross-section of parts of two adjacent mesophyll cells
at high magnification, epi, epidermal cell; meso, mesophyll cell; chl, chloroplast. Bars:
A, 5/im; B, 1 ^m.
Cytoplasmic streaming in Vallisneria
397
epi
"V
meso
8A
#
Fig. 8
398
5. Takagi and R. Nagai
• • • >
epi
meso
9A
cw
chl
ml
cyt
'•
K*
B
Fig. 9
Cytoplasmic streaming in Vallisneria
399
EGTA. These facts may be interpreted as showing that calcium accumulates in the
cytoplasm during irradiation and that an increase in Ca2+ concentration in the
cytoplasm inhibits streaming.
Calcium in the cytoplasm
To demonstrate the increase in Ca2+ concentration visually, specimens were fixed
in the presence of potassium pyroantimonate, which is known to be a fairly specific
precipitant of calcium.
First, wefixedcells just after the pretreatment. In these the cytoplasm did not show
any sign of streaming. Precipitates, as shown in Fig. 8, are formed in the cytoplasm
and the middle lamella of the cell wall. Precipitates are also abundant in the vacuole.
Next, we fixed cells treated with IOITIM-EGTA solution for 25-40min to induce
streaming in all cells. Fig. 9 shows a section of these cells. Only a small amount of
precipitate is seen in the cytoplasm. A few precipitates are observed in the middle
lamella, the chloroplasts and at the border between the cytoplasm and the cell wall.
Precipitates in the vacuole are slight.
On the other hand, precipitates are abundantly visible in the cytoplasm in cells
treated previously with EGTA and subsequently irradiated with far-red light in APW
(Fig. 10). Precipitates accumulated in the chloroplasts (Fig. 10B), mitochondria and
endoplasmic reticulum (data not shown). The middle lamella was heavily stained
(Fig. 10B).
The presence of calcium in these precipitates was confirmed using an X-ray
microanalyser. Arrows a, b, c and d in Fig. 11 indicate the precipitates on which
analysis was done. Four micrographs in the lower portion of this figure show the
results of the analysis alphabetically. Clearly, calcium was present in each precipitate.
Osmium comes from the fixative OsO4 and copper from the grid.
These observations reveal that the intracellular calcium concentration is much
lower when the cytoplasm is involved in streaming than when it is immobile.
Fig. 9. Intracellular calcium deposits in cells treated with lOmM-EGTA for 25-40min
before fixation. Only a small amount of precipitate is observed in the cytoplasm. A few
precipitates are observed in the middle lamella (ml), chloroplasts (chl) and at the border
between the cytoplasm (cyl) and the cell wall (ctv). The sections were not stained, A.
Cross-section of cells at low magnification; B, cross-section of cells at high magnification.
Bars: A, lO^ni; B, 1 fim.
Fig. 10. Intracellular calcium deposits in cells treated with EGTA and then irradiated
with far-red light in APW before fixation. Precipitates are abundant in the cytoplasm and
chloroplasts, and also in the middle lamella. The sections were not stained, A. Crosssection of cells at low magnification. The asterisk indicates a cell killed by cutting with a
razor during the pretreatment of the specimen, B. Cross-section of cells at high magnification, epi, epidermal cell; meso, mesophyll cell. Bars: A, 10^m; B, l^m.
Fig. 11. X-ray microanalysis of precipitates. The electron micrograph shows parts of two
adjacent mesophyll cells from the specimen shown in Fig. 10. Arrows a, b, c and^/ indicate
the precipitates analysed. The results correspond alphabetically to each micrograph shown
in the lower part of the figure. All micrographs show that calcium (CA) is contained in each
analysed precipitate. Omsium (OS) comes from OsO* and copper (CU) from the grid.
400
S. Takagi and R. Nagai
.1...
Fig. 10. For legend see p. 399.
Cytoplasmic streaming in Vallisneria
1I.24KEU*
0.08KEU
Fig. 11. For legend see p. 399.
401
10.24KEU)
402
S. Takagi and R. Nagai
DISCUSSION
All the pretreatment procedures used were needed to cause complete stoppage of
streaming in all cells, in order to obtain a homogeneous preparation of cells as starting
material for the experiments. For example, in the case of a single specimen mounted
immediately after cutting and evacuation, omitting the stage of keeping it under the
original light conditions of the 12 h dark and 12 h light scheme, led to about 10% of
the observed cells exhibiting streaming even after 12-18 h dark treatment. In the case
in which many pieces, say 20, were floated in a glass vessel under the same conditions
streaming was active in almost all cells. And even after complete pretreatment, direct
touching of a specimen with fingers or forceps could induce streaming in the cells.
Therefore, each specimen had to be kept first under the original light condition for
24 h after cutting and evacuation, and then mounted on a glass slide before the last
dark treatment. Some unknown substance(s) that induces streaming seems to be
secreted from the injured end of the leaf or even upon direct touching of the cells.
Further study is needed to identify such a substance(s) and to investigate how streaming is induced, i.e. through decreasing calcium concentration in the cytoplasm or by
another mechanism.
The present study clarifies the microfilament organization in Vallisneria mesophyll
cells. The microfilaments in Vallisneria are known to be composed mainly of F-actin
(Yamaguchi & Nagai, 1981). And because streaming is inhibited by cytochalasin B
(Ishigami & Nagai, 1980), they are supposed to act as a motile apparatus for streaming
in Vallisneria cells. Their configuration, localization and distribution were the same
when the cytoplasm was streaming or quiescent. Therefore, morphological changes
of the microfilaments are not directly responsible for the induction or cessation of
streaming.
In the experiments showing that the induction and cessation of streaming could be
controlled alternately with EGTA and irradiation with far-red light (cf. Fig. 7A, B),
examination of about 50 cells showed that the direction of the cyclosis, i.e. clockwise
or counter-clockwise, did not change after the second induction with EGTA. An
earlier report (Ishigami & Nagai, 1980) suggested that the microfilaments were rather
labile but this statement has not been supported by the present observations. This is
probably due to the short duration of the treatment, i.e. 12-18 h in the dark or about
2h under irradiation, or else it may be due to the fact that the bundles of microfilaments or the microfilaments themselves are unstable in the presence of cytochalasin
B in Vallisneria mesophyll cells, although in characean cells the microfilament bundles remain normal after treatment (Bradley, 1973; Williamson, 1978).
The effect of calcium-chelating agents, EDTA or EGTA, on the velocity of streaming has been examined. Forde & Steer (1976) observed activation of streaming in
Elodea leaf cells by 0-lmM-EDTA and its inhibition by higher concentrations,
10 mM and 100 min. They confirmed further that active streaming was inhibited by
external application of Ca2+, and streaming inhibited by a higher concentration of
EDTA returned to normal when Mg2* was applied externally. Yamaguchi & Nagai
(1981) reported that streaming in Vallisneria epidermal cells could be induced by the
Cytoplasmic streaming in Vallisneria
403
application of 5-10 mM-EGTA. Although these observations were made under white
light, they stressed that Ca2+ has a significant effect on secondary streaming. The
present study, which was performed with complete elimination of the light effect,
confirmed the induction of cytoplasmic streaming by lowering the calcium concentration in the cytoplasm.
The stimulating effect of EGTA on streaming is rapid (Fig. 2), which is readily
plausible if changes in a low Ca2+ concentration in a small cytoplasmic compartment
and a low rate of calcium transport from the vacuole are involved. Though vacuolar
Caz+ concentration is supposed to be rather high during treatment with EGTA (cf.
Fig. 8), a lack of precipitates in EGTA-treated cells was observed (cf. Fig. 9). This
may be due to Ca2+ chelation in the free space by EGTA, unavoidably carried over
into the fixative and given access to the cell during fixation, and/or to Ca2+ chelation
by EGTA penetrating into the vacuole during the treatment.
The exact concentration of calcium in the cytoplasm involved in streaming could
not be determined in the present study. According to the observations on characean
internodal cells, Ca2+ concentration in the streaming cytoplasm is about 10~ 7 M and
streaming is inhibited at a higher concentration (Williamson, 1975; Williamson &
Ashley, 1982; Tominaga & Tazawa, 1981). Therefore, it may be reasonable to suppose that the Ca2+ concentration in the cytoplasm during normal rotational cytoplasmic streaming in Vallisneria mesophyll cells is also maintained at about 10~7 M or less.
Inhibition of streaming by irradiation with far-red light occurred more rapidly
when calcium was present in the external medium than when it was not (Fig. 6). This
is attributed to the increase in calcium concentration in the cytoplasm. However, little
is known about how such an increase in calcium transport into the cytoplasm is
influenced by far-red light. Calcium accumulation in cells of Mougeotia has been
reported to be accelerated by irradiation with red light and this effect is cancelled by
subsequent irradiation with far-red light (Dreyer & Weisenseel, 1979). Efflux of
calcium from oat coleoptiles and their protoplasts is enhanced by irradiation with red
light, and subsequent irradiation with far-red light induces a decrease to levels near
the dark control (Hale & Roux, 1980). Examining the effect of red light on the
induction of streaming and its relation to that of far-red light may clarify whether a
pigment such as phytochrome plays a role in calcium transport in Vallisneria.
The precipitant pyroantimonate can penetrate the biological membrane and hence
can be used to fix Ca2+ in biological tissues. It is also useful as an electron-microscopic
cytochemical reagent because it forms a dense precipitate with calcium (Caswell,
1979). As pyroantimonate can react with Mg2"*" and Na + , as well as Ca2+, to cause
precipitation, deposits are not always precipitates of Ca2+. However, X-ray
microanalysis revealed that calcium was present in each precipitate even though added
magnesium and/or sodium were coprecipitated. The original X-ray microanalysis
data showed the coexistence of antimony and calcium in the precipitates. Fig. lla,
b, c, d presents the results obtained by computer analysis in which the fraction
containing antimony was subtracted from that in the original micrograph.
On the basis of these observations, we conclude that rotational cytoplasmic streaming in Vallisneria cells can be induced when the calcium concentration in the
404
S. Takagi and R. Nagai
cytoplasm decreases and the induced streaming stops when calcium concentration in
the cytpolasm increases.
We thank Professor emeritus N. Kamiya of Osaka University for his valuable criticism, and also
to Professor H. Shibaoka for his profitable suggestions. We are indebted to Dr N. Matsumoto for
lending out the photodiode detector to measure the intensity of light, and to Mr Saitoh (Naka
Wooks, Hitachi Ltd) for performing X-ray microanalysis.
This work was partly supported by grants-in-aid from the Japanese Ministry of Education,
Science and Culture.
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(Received 4 January 1983-Accepted 21 February 1983)