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1.
Introduction - why Vaucheria for phototropism study?
I
would like to introduce some recent progress in studies of phototropism
and relating bluelight responses in a coenocytic alga, Vaucheria.
Before going into the subject, however, we probably need a brief introduction
to historical background of phototropism and the significance of the use
of Vaucheria for the study of phototropism.
Of
many behavioral responses of plants and fungi to environmental vectors
phototropism is the most popular phenomenon and hence most extensively
studied. Even high school students know that Darwin (1880) was the first
man who studied the phototropism of gramineous coleoptiles in modern sense
of science. The most important point of his finding is that the apical
tissues of the coleoptile is the sensor of the light vector and it dispatched
some signals towards the basipetal target tissues that produce the curvature.
Boysen-Jensen (1910) found these inner signals to be water-soluble substance(s)
that was transmitted through an inserted gelatin layer. Went (1928) developed
the famous Avena curvature test to bioassay this substance; and
this substance was later named auxin by Koegel and Haagen-Schmit
(1931).
However,
it was not until 1948 when the auxin was confirmed to be indole acetic
acid (IAA) by Wildman and Bonner with colorimetric method and 1959 when
Shibaoka and Yamaki chromatographically identified. Having this nice model
system, not only studies of ptotropism of higher plants have flourished
but plant physiology have prospered.
We
have to remind, however, that many efforts have also made since the end
of the last century to look for much simple model system. In the previous
issue of ISK series (No. 4) Ootaki reviewed studies of phototropism in
Phycomyces in some detail. I have also developed physiological
analyses of phototropism in Vaucheria since 1975 when I was a graduate
student in Osaka University.
Vaucheria is a chl. a and c containing coenocytic tip-growing
alga. It belongs to Xanthophyceae, Chrysophyta, Stramenopile (Chromista).
Stramenopile is a large Kingdom including also brown algae, bacillaria,
chrysophytes, and even oomycetes. The body of Vaucheria consists
of sparsely branching tube of diameter ranging from 30-130 ｵm (that of
mainly used V. terrestris sensu Goetz is 50-70 ｵm).
At
the apex of each branch typical tip growth occurs at the rate about 200
ｵm h-1. Phototropic response from perception of intensity and direction
of light to manifestation of bending all occurs within narrow apical region
of the cell. Comparing to many other tip-growing cells, such as young
sporangiophores of Phycomyces, protonemes, or pollen tubes, Vaucheria
and algal coenocytes are convenient and suitable materials because they
continue active tip-growth for long periods.
The
word phototropism (Phototropie) was first used by Oltmanns in 1892
when he described the positive and negative phototropism of Vaucheria.
Before
him, even by Darwin (1880), the bending response of plants to the light
was called heliotropism. Since the response is not to the sun, but to
the flux of photon, phototropism is much better terminology. However,
the true reason why Oltmanns renamed heliotropism was not simply that
he was genius but probably based on his careful observation of the nature.
He first observed that Vaucheria bent towards weak light but away
from intense sunlight.
One
may know a priori that coleoptiles, hypocotyles and stems of most
terrestrial plants always show positive phototropism even when they are
unilaterally irradiated with intense sunlight. Adaptation to strong sunlight
is no doubt an indispensable trait for terrestrial plants and fungi to
survive under direct sunlight.
There
is only few exception, besides Vaucheria, among plants and fungi
that has the ability to change the sign of phototropism in response to
the light intensity. (An exception is a tropical Araceae Monstera gigantea
seedling: The twining plant bends and reaches a dark host tree at high
intensity of light; and a shaded host tree at lower light intensity (p.
531 in Mohr and Schopfer 1992). The ability of Vaucheria to change
the sign of phototropism can also be observed in the natural habitats.
It seems to be ecologically very important to optimize photosynthesis
and for the alga to move away from harmful strong sunlight.
The principle of the mechanism of detection of light-direction which is
necessary for phototropic response is to compare number of photons absorbed
by photoreceptors at the near and far sides of the organ (One instance
mechanism). When the light intensity is very high, the photoreceptors
at the both sides are saturated and consequently no bending is resulted.
Phototropic indifference seen in most land plants to strong unilateral
light can be explained by the saturation.
What
is then the necessary condition for the light intensity-dependent phototropic
inversion in Vaucheria? The simplest explanation is that there
is two different photoresponses specific for positive and negative bending
and that they have different threshold levels and different kinetics.
There, one of the elemental steps of negative phototropic response may
be regulated by another light response for the positive phototropism.
Although
we have not yet any substantial evidence for this view, we have recently
obtained an evidence that the positive to negative switch mechanism is
closely related with the cytoplasmic level of free Ca2+. We are postulating
a hypothesis of the involvement of bluelight-induced influx of calcium
ion in the regulation and inversion of phototropic bending of Vaucheria.
This will be dealt with in the following sections.
I
have made basic physiological analyses of phototropic responses of Vaucheria
since 1975. See Reference section for old papers of phototropism
of Vaucheria before 1987 (1975 a, b; 1977 a, b; 1979; 1980; 1981a;
1982). Kataoka's studies on growth and phototropism before 1979 have been
reviewed by Briggs and Blatt (1980). In this issue I would like to introduce
papers published after 1987. For further details some recent reviews of
Dennison (1979, 1984) and of Pohl and Russo (1984) will be useful.
Also,
Wada and Kadota (1989) and Dring (1988) nicely reviewed some recent studies
on phototropism and photomorphogenesis in lower green plants. They
overview the present status of the phototropism studies and suggest problems
and future approaches. For Japanese students Kataoka's
review (1981b, 1991, 2001) may
also be useful to get information not involved in this issue.
2.
Light-growth response as a conditio sine qua non of the phototropism
in the tip-growing alga Vaucheria.
Positive
phototropic bending of Vaucheria is initiated by a local acceleration
of tip growth at the flank of the hemispherical apical dome (Kataoka
1975 a, b). This process is microscopically detectable as a quick shift
of the apical transparent cap region towards the flank of the apical dome
and subsequent hump formation at the point. This mechanism, called
bulging, is a common mechanism of many tip growing cells, such as
fern and moss protonemata, fronds and rhizoids of Boergesenia (Etzold
1965; Jaffe 1960; Ishizawa and Wada 1979 a, b; Iseki et al. 1995 b, Iseki
and Wada 1995).
Some
other tip growing cells, however, bend through differential growth; in
these cells growth zone extend more or less to sub-apical regions and
the side of the faster cell wall extension becomes the convex side. This
type of bending mechanism, called bowing, is seen in the frond
of a marine Chlorophycean coenocyte, Bryopsis (Iseki et al. 1995a),
stage I sporangiophores of Pilobolus (Page and Curry 1966) and
in the gravitropic response of Chara rhizoids (Sievers and Schroeter
1971).
Bulging at the growing apex of Vaucheria starts as quickly
as 2 min after the onset of unilateral bluelight. An actively growing
apex serves also as the steering apparatus. Curvature towards bluelight
source can be detected 3-4 min after the onset of light when the transient
local acceleration of tip growth ceases. The local acceleration of tip
growth at the very tip region also occurs when the apex was uniformly
irradiated with bluelight. Note, however, that in this case instead of
bending a transient acceleration of straight growth takes place. The light-induced
transient growth promotion is called positive light growth response
(LGR). If transient growth retardation is brought about by light,
it is called negative LGR. The LGR does not always mean
net enhancement or inhibition of growth. The growth acceleration is frequently
followed by a long lasted small growth retardation.
Blaauw (1914, 1915,
1919) analyzed the LGR of Avena coleoptiles, Phycomyces
sporangiophores and many other plant organs and concluded that positive
phototro-pism was simply explained by differentially occurring LGRs at
the lighted and shaded sides of the cells or organs. As to positive phototropism
of Avena coleoptiles, the positive bending is caused by decreased
growth rate at the lighted side and the concomitant increase in growth
rate at the shaded side. It is clear that theory cannot explain what is
occurring in Avena coleoptiles.
Blaauw's simple
conclusion was later quantitatively examined and disproved by many researchers.
The most important point for the claim is that LGR is merely a transient
response whereas the phototropic bending continues for hours at fairly
constant rate (e.g. Cholodny 1933).
In the bulging mechanism
of tip growing cell, like Vaucheria, however, the phototropic bending
does not necessarily require continuous acceleration of local growth.
Kataoka (1987) compared intensity ranges, time courses and pH dependencies
of the positive LGR and positive phototropism of Vaucheria and
concluded that the positive LGR was the indispensable condition (conditio
sine qua non) of the phototropic response.
The paper (Kataoka
1987) demonstrated that the pH dependencies of both positive phototropic
response and positive LGR were very similar. Particularly, the photosensitivity
of the LGR showed perfect pH dependency in physiological pH range:
i. e., the threshold at pH 7.5 was 100 times higher than that at pH 5.5.
This indicated that the initial photochemical event of bluelight transduction
closely coupled with proton motive force or with proton fluxes through
plasmalemma at the apex.
3.
Bluelight-induced current influx at the growing apex of Vaucheria
cell, the initial event of the positive light-growth response.
Not
only LGR but also steady-state growth rate in dark or low fluence-rate
light conditions is pH sensitive. The optimum pH of Vaucheria terrestris
sensu Goetz for the growth was found to be 7.0 to 7.5. If the pH was changed
by ｱ0.5 to 3 units after being stabilized in solutions of various pH for
several hours, the growth rate shifted to a new value within 20 min. The
alga more quickly accommodates itself to the new pH, if the shift is small
and is towards optimum pH (7.0-7.5). Width of the cell is also pH dependent:
average diameter (in ｵm) of the 20 tubes measured just below the equator
of the apical dome were 78.3, 76.9, 65.4, 54.7, 54.7 at pH 4, 5, 6, 7,
8, respectively (unpublished data).
At
pH 8.0 the growth rate was the highest, the apical dome was a bullet-shaped
rather than hemisphere, and the tube became slender. Increase in Ca2+
concentration (up to 10 mM) in the bathing solution did not alter the
width of the cell. Thus, it is probable that protons are involved in width
control.
Tip growth is maintained by continuous supply of exocytotic vesicles
to the apical region of the dome and subsequent active exocytosis through
limited area of the apical plasmalemma (Kataoka 1981, 1982). At pH 7.0,
optimum for tip growth, the apical transparent cap, the pack of exocytotic
vesicles, is the largest, extending up to the apical 1/3 of the hemispherical
dome. Since the positive phototropism of Vaucheria is initiated by a rapid
shift of the maximum exocytosis to bluelight-irradiated flank, the exocytotic
apparatus may favor neutral to slightly alkaline pH.
This
is also deduced from bluelight-induced depolarization. The membrane potential
(central vacuole to the external solution) was about -120mV in the external
solution of pH 7.0 under dim green or red light or in darkness; upon irradiation
with bluelight a transient depolarization started after a very short lag
time (unpublished data). The depolarization reached maximum about 50 mV
after 1min, but the potential came back again to the original value after
another 1 min even with continuous bluelight. The depolarization decreased
to about 20 mV when the external pH was 5.0.
Another
electrophysiological approach for the detection of earliest intracellular
signal from the growing apex of Vaucheria was made using vibration
electrode technique in collaboration with Weisenseel in Karlsruhe, Germany
(Kataoka and Weisenseel, 1988). This technique was developed by Jaffe
and Nuccitelli (1974) and used for detecting small extracellular electrical
current which was produced by living organisms (Blatt and Weisenseel,
1980; Blatt et al. 1981; Behrens et al. 1982).
If
a single cell or organ is spatially polarized by either external of inner
factor, the surface area is differently charged. If the cell or the organ
is surrounded by aqueous solution, ions of the solution carries the charges
to neutralize the polarization, and hence formed a current loop traversing
the organ or the cell. With a small platinum-black spherical electrode
vibrating in one plane with audio frequency a small voltage drop between
the two extreme points of the vibration is detected with lock-in-amplifier.
When
the specific resistance of the basing solution is ρ(Ohm
cm), the voltage deference V between distance d (cm) of
the two extreme positions of the vibrating probe is then converted to
current density J (A cm-2) by following equation, according to
Ohm's law :
J=V/ρd
A
large current influx was observed at the growing apex of Vaucheria
(Kataoka and Weisenseel 1988). The current influx took place at the apex,
so far as the alga grew actively. However, the value of the steady current
varied widely. Upon bluelight irradiation the influx always increased
after a few sec of lag time. Red light did not increase the influx. The
bluelight-induced component of the current influx (BLCI) was therefore
analyzed further in detail.
The
BLCI sometimes started after a lag time of 10-20 s, but always
in advance of the start of positive LGR. The BLCL also showed an
oscillation: the maximum influx was after about 3 min, and the second
maximum was found after another 5 min. The BLCI was induced by a 2-sec
pulse of bluelight, which was too small to trigger LGR. Thus, the BLCI
could be the reflection of a primary biological event leading the LGR
and positive phototropism.
As
to the question what ion carried the influx of positive charge (or efflux
of negative charge in the same sense), we do not still have concrete idea.
Proton influx could by the answer, considering that optimum pH for the
BLCI is 6 not 7 (because, the more external protons, the larger influx
is expected). Influx of calcium ions could not be the main component of
the BLCI, because the depletion of external Ca2+ rather increased the
BLCI.
Organic
Ca-channel blockers, nifedipine or verapamil increased the
BLCI too, instead of decrease. Interestingly, however, they canceled refractory
phenomenon of the BLCI and positive LGR that was frequently observed by
the repeated pulses of bluelight. That the BLCI and LGR repeated tirelessly
in the solution containing verapamil (Kataoka and Weisenseel 1988) led
me an idea: bluelight-induced calcium influx may be involved in the in
the positive/negative inversion of phototropic response.
4.
Involvement of calcium in the inversion from positive to negative phototropism
in Vaucheria.
As described above, Vaucheria
has the ability to change the phototropic bending from positive to negative
direction when the light intensity exceeds a critical value. The critical
light intensity varies species and/or strain and also probably with physiological
status.
The
fresh water species, Vaucheria terrestris sensu Goetz, mainly used
as the experimental material bends away from white light of about 6 Wm-2;
this is the lowest value among other Vaucheria species. To experimentally
induce negative phototropism of the most sensitive V. terrestris sensu
Goetz strain, however, at least several hours of irradiation with monochromatic
bluelight from ordinary light source is required. This has made physiological
analysis of negative phototropism impractical for long.
Recently,
however, Kataoka (1988) found that the alga bent away from the unilateral
bluelight source of the intensity only about 2 Wm-2, if the apex was simultaneously
irradiated with strong blue or green light. He then developed the simultaneous
background irradiation method and analyzed the negative phototropic
bending by changing wavelength, intensity, and duration of the background
light, keeping wavelength and intensity of the unilateral bluelight constant.
From now on we sometime use more precisely fluence rate instead
of light intensity, and fluence for the amount of photons (= intensity
x time).
The
negative bending induced by the method with supporting background blue/green
light was strongly and specifically dependent on the external concentration
of Ca2+. Other divalent cations were either toxic or ineffective. If either
the fluence rate of the background blue/green light or the concentration
of Ca2+ was low, only positive curvature was produced. Magnitude of the
negative bending was proportional to the product of fluence rate of the
background light and external calcium concentration. This indicated
that bluelight opened Ca2+ channels of the apex and that resulted elevation
of intracellular level of Ca2+ led the inversion of phototropic bending.
Requirement
of very low, but not nothing at all, Ca2+ also for the induction of positive
phototropic response was strongly suggested by following facts:
1) if external concentration of Ca2+ was between 1 mM and 1 ｵM, only positive
phototropism
was resulted, even when the simultaneous background blue/green light was
very strong.
2) The positive curvature and the tip growth itself were arrested when
Ca2+ concentration
was below 1 ｵM.
3) The background light started 3 min after the unilateral bluelight did
not produce negative
bending any more.
4) Coexistence of high Ca2+ (4.4mM) and the background irradiation 10-fold
sensitized the
positive phototropism.
5) Replacement of Ca2+ by Sr2+, neither positive nor negative bending
was elicited, although
normal tip growth was maintained.
The
paper (Kataoka 1988) was thus the first report that demonstrated the in-volvement
of bluelight-induced Ca2+ influx and eventual elevation of cytoplasmic
level of Ca2+ in the transduction and regulation of the direction of phototropic
responses.
5.
Further evidence of the rise of cytoplasmic Ca2+-level for the regulation
of positive and negative phototropic response.
To
prove the Ca2+ influx through bluelight-gated channels some experiments
with channel blockers and ionophores were conducted (Kataoka 1989, 1990).
If only 2 ｵM LaCl3 was added to the bathing solution that contained 4.4
mM Ca2+, the supporting background irradiation (diffuse strong blue-green
light) did not induce negative bending from the unilateral low fluence-rate
bluelight.
Considering
the impermeability of La3+ through the plasma membrane and the fact that
2,000th fewer La3+ could well cancel the effect of Ca2+, the effect of
La3+ is attributable to closure of Ca2+ channels, not to competition at
the binding site with Ca2+. Inhibition of the negative phototropism by
La3+ was also observed under conditions where the alga was irradiated
with unilateral bluelight for 1 week.
In
contrast to control alga that turned back from bluelight source at 6 W-2,
in the presence of 20 ｵM La3+, it grew straight towards the nearest wall
of the dish where the bluelight intensity was 9 W-2 (Figure 4 of Kataoka
1990).
Organic
Ca2+ -channel blockers, verapamil, nifedipine and nitrendipine
caused nullification of the phototropic inversion also. The last two drugs
are specific blockers of L-type (depolarization-triggered) Ca2+ channels.
Verapamil is thought to bind also with L-type channel, but at the
different site and with a different manner: i. e., it seems to bind to
the inner cavity of the open channel to inactivate it.
The
effect of verapamil is, therefore, expected to occur only after
the channels has been opened by bluelight stimulus, and this was proved
to be the case (Kataoka 1990). The fact that the effectiveness of these
different types of channel blockers were very similar strongly suggests
the presence of L-type Ca2+ channels in Vaucheria. The bluelight-induced
transient depolarization (see section 3) may trigger the opening of the
Ca2+ channels at the apex of Vaucheria.
The
addition of a Ca2+ ionophore, A23187, without supporting background
irra-diation gave very interesting results. The positive phototropic curvature
to a 5-min-pulse of unilateral bluelight (456 nm, 1.7 Wm-2) increased
with increasing concentration of A23187 between 0.1 and 1 µM; however,
the curvature to a 10-min-pulse of the same light source decreased with
increasing concentration of A23187 and finally showed slightly negative
curvature at 1 µM (see Figure 7 of Kataoka 1990). This indicates:
1) as suggested above, if Ca2+ has previously been introduced into the
cell (through
membrane holes artificially made by the ionophore), the unilateral bluelight
can more
quickly raise the cytoplasmic level of Ca2+ at the lighted side of the
apical dome,
and hence the larger positive bending;
2) if the Ca2+ level has already raised by A23187, additional larger fluence
(10 min) of
unilateral bluelight quickly elevates Ca2+ level above supraoptimum level,
and this leads
inversion of phototropism.
The
action of Ca2+ channel blockers and A23187 is schematically summarized
in Figure 8 of Kataoka (1990). The counteracting effects of the drugs
are explained by the shift between low and high cytoplasmic Ca2+ levels.
The diagram also predicts that the fluence-response curve for positive
phototropism under safe red background light will soon or later turn to
decrease and goes into negative at the large fluence region, if a very
strong unilateral bluelight is practical.
6.
Use of a high-power continuous-wave argon-ion laser for induction of negative
phototropism.
Although
the simultaneous background irradiation method provides a good
simulation of the negative phototropism observed in the natural habitats,
a much simpler method, namely, strong unilateral bluelight irradiation
alone, without any supporting background light, would be far better for
kinetic analysis of the regulation of the direction of phototropic bending.
Kataoka
and Watanabe (1992, 1993) investigated this approach using a high power
continuous-wave argon-ion leser (Innova 20, Coherent, Palo Alto, 457.9
nm) as a source of single very strong bluelight. When the beam was expanded
to 6 mm in diameter, fluence rate at the specimen level reached 5.3 kWm-2.
The
growing apex of Vaucheria terrestris sensu Goetz bent positively
to the unilateral blue laser beam when the fluence rate was below 60 Wm-2,
irrespective of the solution contained either 0.4 mM or 4.4 mM Ca2+. The
positive bending obeyed reciprocity law until the fluence reached
19 kJm-2 (53 Wm-2 x 6 min), as was the case in experiments using ordinary
incoherent light source. However, when the fluence rate became higher
than 60 Wm-2 and duration of irradiation was between 10 and 300 sec, the
positive curvature decreased.
The
alga finally showed a deep negative curvature when either the fluence
rate or the duration of irradiation was further increased. The decrease
in positive curvature and the development of negative phototropic response
was greatly enhanced in the presence of 4.4 mM Ca2+, and did not obey
reciprocity law.
The
mechanism that determine the sign of phototropism seemed to require at
least several sec of irradiation, even when the fluence rate was sufficiently
high. Then, on which parameter, fluence rate or duration of irradiation,
does negative phototropism depend more strongly? Comparing relationship
between log fluence rate and curvature with that between log irradiation
time and curvature (Figure 5 of Kataoka and Watanabe 1993), the switch
over mechanism seems to be dependent more strongly on fluence rate not
on the duration time, especially fluence rates between 60 Wm-2 and 600
Wm-2.
The
log fluence rate-response curves clearly indicates that the algal cell
apex measures the fluence rate of the unilateral bluelight during the
period between 10 and 100 sec after the onset light. The alga will then
compute the next direction and magnitude of bending. This is also supported
by the microscopic observation of the apex during the response: upon unilateral
laser irradiation, the apical transparent cap region first moves towards
the light source until 4-5 min, then turns back after 6 min to the shaded
side.
7. Other environmental factors modifying
thepositive/negative
phototropism.
As
discussed above, pH of the surrounding medium greatly affects the tip
growth and phototropism. Since phototropic response of a tip-growing cell
can be taken as modified cellular polarization activities, the low pH
syndromes, such as broaden growth region, expansion of apex, low exocytotic
activity and low photosensitivity may be the result of short circuited
apicobasal polarity generated by transcellular transport of proton.
There
are some evidences for localized proton pumping out activity at basal
part of organs or cells and also localized passive current influx at the
growing apices (see e.g. Nuccitelli, 1986). This may vice versa indicate
that bluelight strengthens cellular polarity via stimulation of either
proton pump activity or channel opening.
The
phototropic inversion of V. terrestris sensu Goetz have recently
been found to be stimulated by 50 - 150 mM NaCl (unpublished data). The
alga shows negative phototropism from bluelight of only 3 Wm-2. Since
the same effect is observed with the sorbitol solution having the same
osmotic pressure, NaCl seems to act simply osmotically: namely, since
V. terrestris sensu Goetz cannot take up both NaCl and sorbitol,
its turgor pressure cannot be restored in the salt or sorbitol solution.
Thus, the decrease in turgor pressure is hypothesized to be coupled with
the enhancement of phototropic inversion.
This
view was supported by comparing the effects of NaCl and sorbitol of the
same osmotic pressure with the brackish species, V. dichotoma.
While fresh water species, V. terrestris sensu Goetz, cannot regulate
its turgor pressure, V. dichotoma has an ability to keep its turgor
pressure constant unless the external osmotic value exceeds 300 mOsm (Henschel
et al. 1991, Kataoka unpublished data).
Surprisingly,
V. dichotoma showed positive phototropism when the external osmotic
pressure was raised with 0 - 150 mM NaCl, while it showed negative bending
when NaCl solution was replaced by isotonic sorbitol solution. This difference
can be explained by the ability of turgor regulation. Vaucheria dichtoma
can take up NaCl to increase its osmotic pressure and restore its turgor;
on the other hand, since the algae cannot take up impermeable sorbitol
nor synthesize any other organic osmotica, its turgor pressure cannot
be restored.
It
is possible that the loss of normal turgor is linked with phototropic
inversion through depolarization and/or elevation of cytoplasmic level
of Ca2+. To elucidate the causal relationship of these factors, combined
physiological analyses of osmotic/ionic regulation and phototropism are
now being conducted (cf. Nakagawa et al. 1974, Kataoka et al. 1979).
8.
Bimodal polarotropism
Vaucheria
terrestris sensu Goetz shows novel polarotropism. Bending response
to the direction of E-vector of the linear polarized light
is defined as polarotropism (Büning and Etzold, 1958, Etzold, 1965,
Wada and Kadota 1989, Dennison 1979). Polarotropism has frequently been
observed in fern and moss protonemata. The tip growing protoneme cells
bend perpendicularly to the E-vector of polarized red light
whereas the rhizoid cells bend parallel to the E-vector.
Their spatial (angular) relationship does not change by the change in
light intensity. Polarotropism is a clear example of action dichroism.
Since
flavins, carotenoids and many other pigment molecules are dichroic, having
prefer E-vector orientation for the light absorption, detection
of action dichroism in the system is an indication of these molecules
being oriented parallel to each other in a fixed array. Since plasmalem-ma
and cortical cytoplasmic layer are the most stable two-dimensional structures
of plant cells, the dichroic orientation of pigment molecules is an evidence
for their localization in plasmalemma or outermost layer of the cell (Dennison
1979).
When
thalli of V. terrestris sensu Goetz growing in Petri dishes were
continuously illuminated from above with polarized white light, some of
apices grew perpendicularly to the E-vector, but the other,
parallel to the E-vector (Kataoka et al. 2000). The proportion
of apices growing parallel to the E-vector increased with
increasing intensity of the polarized light. At a moderate light intensity,
the algal filaments showed a cruciform mat. Other species, such as V.
sessilis and V. dichotoma, which had higher phototropic inversion
intensity, grew predominantly normal to E-vector of the
polarized light.
This
novel cruciform growth of V. terrestris sensu Goetz is apparently
closely connecting with its high ability of positive/negative phototropic
inversion. The localization of photoreceptor molecules has been assumed
to be at the outermost layer of the apical hemisphere, from results of
microbeam irradiation experiments (Kataoka 1975b, 1980).
Since
the apical 1/3 area of the hemispherical apical dome is the site of photoreception
and bulging, polarized light whose E-vector is right angle
(90 degrees) to the cell axis must be maximally absorbed by the photoreceptor
molecule at the extreme apical area. If the intensity of the polarized
light exceeds the optimum level, the tip growth becomes to be inhibited
and the bulging occurs at the flank of the hemispherical dome where the
absorption by the photoreceptor molecules is smaller, and consequently
algal filaments growing parallel to the E-vector becomes
predominant in population.
The
cruciform arrangement of the tips indicates the discrimination level for
negative phototropism are varying within a certain range among spatially
separate branches in the coenocytic continuum. From these observation
we proposed to rename perpendicular- and parallel- polarotropism to positive-
and negative polarotropism, respectively.
9. Bluelight-induced branching, a cytomorophogenesis
Bluelight-induced branching is another important
growth-relating photore-sponse of Vaucheria found by Kataoka (1975b).
Irradiation of a narrow basal part of the Vaucheria tube with moderate
intensity of bluelight induced accumulation of chloroplasts in the outermost
cytoplasmic layer of the irradiated region. And at the shortest after
4 h of the onset of irradiation a protrusion arose from the center of
the bluelight-irradiated region.
The
fast occurring chloroplast accumulation was analyzed by Blatt and
his colleagues to some extent (Blatt 1983; Blatt and Briggs 1980; Blatt
and Weisenseel 1980; Briggs and Blatt 1980). They observed an outward
electrical current from the blue-light-irradiated region of V. sessilis
and reported that a reticulation of cortical fibers preceded the chloroplast
accumulation. They correlated the outward current to an activation of
electrogenic proton pump and suggested this as a direct cause of actin
reticulation in the irradiated region.
The
moving chloroplast would be trapped in the irradiated region where the
chloroplast-carrying cortical filaments had been destroyed. However, the
actin nature of the cortical fiber is still not confirmed: although Blatt
et al. (1981) demonstrated actin filaments in cytoplasm squeezed out of
Vaucheria cell, this was not a direct evidence for the disruption
of action filament in the blue-light-irradiated region.
Using
rhodamine-conjugated phalloidin or fluorescent dye-conjugated actin antibodies,
we succeeded to analyze the dynamics of actin microfilaments during the
chloroplast movement (Mineyuki et al. 1995, Takahashi et al. unpublished
data).
In
this issue, however, the chloroplast photoaccumulation is not dealt with
further in detail. It is important to notice that although the chloroplasts
accumulation in the cortical cytoplasmic layer completes 30 -40 min after
the onset of bluelight, the endoplasm that contains numbers of nuclei
and chloroplasts starts to accumulate in the irradiated region 1 h after
the onset of light.
Using
several kinds of inhibitors, we found that accumulation of chloroplasts
is necessary but not sufficient by itself. Accumulated chloroplasts probably
serve as the energy supply for the development of a branch, because DCMU
that does not inhibit chloroplast accumulation completely inhibit the
branching.
On
the other hand, the accumulation of nuclei seems to be indispensable
for the branch induction. By means of immunofluorescence microscopy of
microtubules (MT) and DAPI staining of nuclei we found that every nucleus
has one long MT bundle, 50 - 60 µm in length, at its head and
moves into the bluelight-irradiated region being pulled by the MT bundle
(Takahashi et al. 2001). Very similar nucleus-MT complex has been reported
by Ott (1992) in different Vaucheria species, this unique structure
seems to be common in Vaucheria.
Accumulation of nuclei and MT bundles was detected only 1 h after
the onset of bluelight. Together with nuclei and MT bundles, protoplasm
also flowed into the irradiated region; the thickness of protoplasm increased
with increasing time. Nuclear density in the irradiated region increased
up to 2 fold during the following 2 - 3 h and the MT bundles were concomitantly
broken into randomly orienting short and thin fragments. In the adjacent
shaded region the MT bundles were kept intact.
The
nuclear accumulation and the bluelight-induced branching were completely
inhibited by amiprophosmethyl (APM), a herbicide that decomposes
MT, and by cytochalasin A (CA). APM indeed completely destroyed
the MT bundles; nuclei were eventually aggregated to form flocks in the
cytoplasm. Although CA did not disturb nuclear distribution, nuclei
lost their motility completely, because they lost MT bundles. The both
drugs, however, allowed the accumulation of chloroplasts. Although the
action mechanism of CA must be examined further, the results clearly
indicate that chloroplast accumulation is insufficient by itself for the
induction of branching.
A
large inward current superseded the initial outward current from the bluelight-irradiated
region after about 2 h (Kicherer 1985). The branching occurred only when
this large inward current continued. Taking into consideration that inward
currents are always seen at the actively growing tip of the branch (Kataoka
and Weisennseel 1988), the current influx is probably prerequisite for
the induction of branch primodium from the center of the irradiated region.
This
may also suggests that the site of branching was determined as early as
2 h after the onset of light. Accumulated and bluelight-irradiated nuclei
probably started expression of genes of certain enzymes necessary for
the development of a branch. The inward current may also occur only after
newly synthesized channel proteins have been incorporated in the plasmalemma.
Expression of genes for cellulolytic enzymes by the accumulated nuclei
is also logically predicted.
In
conclusion, we are finding that a quite different process is functioning
in cyto-morphogenesis of coenocytic cells. i. e., gathering nuclei
without nuclear division. In multicellular plants morphogenesis always
require the formation of meristem, i.e., mitosis and cell division. Why
so? We now postulate a principle: for forming a new shape gathering
necessary numbers of nuclei is important, not always is the mitosis.
To
gain necessary amount of nuclei in a narrow region, multicellular organisms
have inevitably to use mitosis. Coenocytic cells, on the other
hand, can attain this quickly and simply by accumulating nuclei from adjacent
area, because they have no septum between nuclei. Accumulation of nuclei
may be much easier than to wait nuclear division. However, this principle
is adoptable only in coenocytes.
Such
approaches will shed light to the role of individual nucleus in the coenocytic
cells.
How is coenocyte organization maintained?
Is monarchy, anarchy, republic or democratic the coenocytic organization?
And what is the merit of being multinuclear coenocyte in the ecosystem
and in the evolution?
These
exciting questions will no doubt lead us to a new discipline, the cell
ecology.
10.
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