Phototropism and photomorphogenesis of Vaucheria.

1. Introduction - why Vaucheria ?
2. Light-growth response as a conditio sine qua non of the
phototropism in the tip-growing alga Vaucheria.
3. Bluelight (BL)-induced current influx at the growing apex of
  Vaucheria cell.
4. Involvement of calcium influx in the inversion from positive
  to negative phototropism in Vaucheria.
5. Further evidence of the rise of cytoplasmic Ca2+-level for
  the regulation of positive and negative phototropic response.

6. Use of a high-power argon-ion laser as the source of very
  strong unilateral BL for induction of negative phototropism.

7. Other environmental factors modifying the positive/
  negative phototropism.

8. Bimodal polarotropism
9. BL-induced branching: a cytomorphogenesis.
10. References


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 :

 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.

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