Temporal control of differentiation by the cell nucleus: evidence for a -specific nuclear suppression of enzymes at the early generative phase in

HORST BANNWARTH

Institut fur Naturwissenschaften und ihre Didaktik, Abt. Biologie, Uniuersitat zu Koln, Gronewaldstr. 2, 5000 Koln 41, Germany and EGON J. DE GROOT*

Max-Planck-Institut fiir Zellbiologie, Abt. Schweiger, Rosenhof, 6802 Ladenburg, Germany * Author for correspondence

Summary

The role of the cell nucleus in controling steps of nuclear action is species-specific, the nucleus of the differentiation has been revealed by comparing species was replaced by a nucleate and anucleate cells of Acetabularia (Berger nucleus of the species . The et al. 1987; Bonotto, 1988). In all cases investigated so foreign nucleus did not suppress the increase in far, events related to the generative phase of the life dCMP deaminase (EC 3.5.4.12) enzyme activity. This cycle are expressed earlier in anucleate cells. Conse- result indicates that the suppression by the nucleus quently, the existence of a nuclear suppressor is species-specific. delaying the onset of generative functions in the intact cell has been postulated (Bannwarth and Key words: Acetabularia, enzyme acitivity, nuclear Schweiger, 1983). In order to determine whether this suppression.

Introduction suppressed by implanting nuclei. This result excludes the possibility that the early onset is a non-specific reaction to The life cycle of the unicellular and uninucleate marine the surgery, and means that the cell nucleus influences the green alga Acetabularia can be subdivided into a vegetat- onset of expression. The exclusion of the cell nucleus ive and a generative phase (Bannwarth, 1988; Schweiger, by other means (ligation, Actinomycin D treatment) 1969). The genetic information is transferred from the always results in an earlier onset of generative events nucleus into the cytoplasm much earlier than needed for (Bannwarth and Schweiger, 1983). gene expression (Hammerling, 1934; Harris, 1970; In order to elucidate the molecular mechanism of the Schweiger, 1980). The duration of the interval between the nuclear control, it is necessary to answer first the question transcription of nuclear DNA and the appearance of the of whether the suppression is species-specific or not. corresponding morphological and physiological features Therefore the activity of dCMP deaminase, an enzyme can be weeks or even months. The genetic program is which is regulated in the early generative phase, has been suppressed until its realization by an as yet unknown investigated (Bannwarth et al. 1982; Schweiger et al. mechanism. 1984). It was discovered many years ago that removing the cell nucleus of an Acetabularia cell leads to an earlier formation of the cap (Beth, 1953; Hammerling and Materials and methods Zetsche, 1966; Werz, 1965). This indicates that the nuclear suppression is removed by enucleation (Rommelaere and Acetabularia was cultured in a synthetic medium as described Hiernaux, 1975; Zetsche, 1966). The rate of total protein before (Berger and Schweiger, 1980; Schweiger et al. 1977). synthesis is also different in nucleate and anucleate cells, Zygotes of a species Acetabularia acetabulum=mediterranea and being higher in enucleated cells (Brachet et al. 1955). Acetabularia crenulata were illuminated and the were These are some indications of the existence of nuclear grown for 60 days. Cells of a medium cap size (diameter: 3—4 mm) control by suppression of gene expression. In addition to were selected for the experiments. morphological characteristics, functional features, in Nucleate cells, anucleate cells and intraspecific grafts were particular enzyme regulation, can be used to characterize pretreated and prepared as previously described (Bannwarth and Schweiger, 1983). Interspecific grafts were made by replacing the distinct parts of the life cycle (Berger et al. 1987; nucleus-containing rhizoid of the species Acetabularia acetabu- Schweiger, 1982; Schweiger and Berger, 1979). lum by a rhizoid of the species Acetabularia crenulata. The early onset of deoxycytidine monophosphate The medium was changed after two weeks and the enzyme (dCMP) deaminase activity induced by enucleation can be activity was estimated every 3-5 days. Enzyme activities of Journal of Cell Science 100, 863-868 (1991) Printed in Great Britain © The Company of Biologists Limited 1991 863 dCMP deaminase (Bannwarth et al. 1982) were estimated in four cells. The incidence of cyst formation and the translocation of from the stalk into the cap were recorded over the 40-45 day duration of the experiments.

Results Intraspecific grafts The activity of dCMP deaminase increased dramatically in nucleate cells of Acetabularia acetabulum after the growth of the cap ceased (Bannwarth et al. 1982; Bannwarth and Schweiger, 1983). The events that led to this regulation occurred some time before the enzyme activity started to increase. This conclusion can be drawn from the fact that enzyme activity also increased in cells which were enucleated two or three weeks earlier. In all the cases investigated so far, the regulation was achieved by de novo synthesis of enzyme proteins, independent of the site of synthesis, on 70 S or 80 S ribosomes (Schweiger et al. 1984). The enzyme activity was not regulated synchronously in nucleate and anucleate cells. Depending upon the stage of development, the increase in enzyme activity occurred suddenly or after a certain lag phase. In each case it was earlier in enucleated than in intact cells. The early increase in enzyme activity in the anucleate cell seemed not to be due to the surgery, but due to the 10 20 absence of the nucleus. In order to prove this assumption, Time (days) the nucleate cells were also subjected to surgery. The cells were subdivided into a larger anterior, anucleate part and Fig. 1. Suppression of dCMP deaminase activity in nucleate cells and in intraspecific grafts of Acetabularia acetabulum. a smaller, nucleate posterior rhizoid part, and sub- Homogenates taken from four cells were incubated at 37 °C for sequently recombined (Bannwarth and Schweiger, 1983). 1 h in the presence of dCTP and [14C]dCMP as described These cells exhibited a delayed increase in enzyme earlier (Bannwarth et al. 1982). Values are given as activity as is typical for nucleate cells. Cysts were also radioactivity in [uC]dUMP per cell. The experiment was formed at the same time in treated and untreated nucleate started with cells of 3 mm cap diameter and terminated with cells (Fig. 1). cells of 11 mm cap diameter. Approximately 50% cysts finally This was in accordance with the hypothesis that the were formed. •, nucleate untreated controls; •, nucleate retransplanted cells (%); A, cysts in the untreated cells (%); A, nucleus normally suppresses all the steps leading to the cysts in the retransplanted cells (%). onset of generative events until its disintegration. The cell nucleus, therefore, not only gave early instructions to the cell cytoplasm, but it also influenced the temporal control after the beginning of the experiment, the influence of the and the sequence of developmental steps. crenulata nucleus became apparent by a dramatic new increase in dCMP deaminase activity in the interspecific Interspecific grafts grafts, while it increased only slightly in the anucleate The next question concerned the species specificity of the controls. suppression described above. It could be answered by It seemed therefore that the foreign nucleus did not replacing the cell nucleus with a foreign nucleus from control the host-specific genetic program of Acetabularia another species. The cell nucleus of Acetabularia acetabu- acetabulum, but expressed its own species-specific regulat- lum cells was removed by cutting off the rhizoid and ory instructions of Acetabularia crenulata at a later time. replaced by the nucleus-containing rhizoid of Acetabularia The species-specific influence of the foreign nucleus on the crenulata. regulation of enzyme activity can be pointed out by The result of this experiment was that the foreign comparing heterologous grafts with the untreated cells of nucleus was not able to suppress the early increase of both species (Fig. 3). The activity of dCMP deaminase enzyme activity. It could not regulate the generative activity started to increase earlier in the Acetabularia functions as the homologous nucleus does. The enzyme crenulata cells than in Acetabularia acetabulum. Again, in activity increased after the exchange of the nuclei at least the hybrid grafts the enzyme activity behaved at first in a as fast as in enucleated cells (Fig. 2). This indicated that similar manner to that in the anucleate cell, showing the the suppression was species-specific. Heterologous nuclei immediate typical small increase, and in addition, 28 days obviously were unable to accomplish the temporal control after the nuclear exchange, the foreign nucleus of the of generative functions in a foreign cell. species Acetabularia crenulata provoked a new species- On the other hand this statement does not mean that the specific increase in enzyme activity. This increase was foreign nucleus has no influence on the regulation of the parallel to that of crenulata cells, but occurred with a delay enzyme activity in the hybrid cells. It took some time of about four days. This time was obviously needed for the before a significant difference between anucleate cells and change from the acetabulum to the crenulata type. cells with a foreign nucleus could be detected. Three weeks This means that a nucleus-dependent regulation in the 864 H. Bannwarth and E. J. de Groot 2.5

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10 20 30 20 30 LO Time (days) 10 Time (days) Fig. 2. Suspension of the suppression of dCMP deaminase activity increase in anucleate cells of Acetabularia acetabulum Fig. 3. Species-specific regulation of dCMP deaminase activity and in cells of this species with a foreign nucleus (interspecific in nucleate cells of Acetabularia acetabulum and Acetabularia grafts, creniXaceto). In the absence of the cell nucleus the crenulata as well as in interspecific grafts of both species. The increase of enzyme activity was not suppressed. A nucleus enzyme activity started to increase two weeks earlier in taken from another Acetabularia species could not substitute Acetabularia crenulata than in Acetabularia acetabulum. In for the homologous nucleus. It was not able to suppress the Acetabularia crenulata the level of enzyme activity was early increase of enzyme activity. Cysts were not formed in elevated prior to cyst formation, while in Acetabularia anucleate cells and in the interspecific grafts. Enzyme activity acetabulum the enzyme activity was concomitantly increased was determined as described in Fig. 1. O, anucleate cells of with the proceeding cyst formation. The heterologous grafts Acetabularia acetabulum; O, nucleate interspecific grafts, exhibited first the early increase, which was characteristic for crenjXaceto hybrids. anucleate cells and later, after 28 days, a second increase, which parallels that of crenulata cells. It was retarded approximately four days relative to the enzyme activity increase in untreated cells of Acetabularia crenulata. Enzyme creni x aceto hybrids could not be detected prior to day 20 activity was determined as described in Fig. 1. 9, nucleate following the nuclear exchange. Until this time the untreated controls of Acetabularia acetabulum; •, nucleate enzyme activity resembled that of anucleate Acetabularia untreated controls of Acetabularia crenulata; •, nucleate acetabulum cells, the acceptor species. Later, the first interspecific grafts, creniXaceto hybrids. Ci, start of cyst acceptor cell-controlled increase ceased until a new formation in Acetabularia acetabulum; C2, start of cyst enzyme activity increase, the crenulata-specific, followed. formation in Acetabularia crenulata. This would mean that the genetic programs of both species, first of Acetabularia acetabulum and later of Acetabularia crenulata, were expressed in succession in the interspecific grafts, and that enzyme activity is could be followed with the naked eye (Bannwarth, 1985a; regulated by both the cytoplasm and by the nucleus Bannwarth, 19856; Bonotto, 1988). The late events in the (Figs 2, 3). cell cycle of Acetabularia were not regulated in the same In addition, this finding is in accordance with the way in both species. While in Acetabularia acetabulum the conclusion that the nuclear-dependent regulation of activity of dCMP deaminase was increased immediately enzyme activity is species-specific. prior to cyst formation, the activity of this enzyme was increased significantly earlier in Acetabularia crenulata Translocation of chloroplasts and cyst formation (Fig. 3). Cysts were formed in this species when the cells Another interesting question is how the regulation of had already attained a high level of enzyme activity. enzyme activity is correlated with other visible changes in In intraspecific grafts, the translocation of chloroplasts cell differentiation. Other functions of the generative and cyst formation were not impeded. The generative phase in Acetabularia, such as the translocation of events were only slightly retarded in surgically treated chloroplasts from the stalk into the cap and cyst formation, cells and were accelerated if the nuclear influence was

Nuclear control of an enzyme in Acetabularia 865 NUCLEAR SUPPRESSION translocation. From these observations one may conclude that translocation and cyst formation are also regulated species-specifically. nucleate control

Discussion operated The experiments presented here, giving new information on the temporal control of differentiation by the cell NUCLEAR SUPPRESSION SUSPENDED nucleus, included a nuclear exchange and an estimation of enzyme activity in the same cell. Until now, this type of experiment could only be carried out with a limited anucleate number of Acetabularia cells, and it is a disadvantage that an appropriate statistical evaluation is not possible. Some differences between single experiments may occur and are within the experimental error of the system. They are temporarily 1 looted admitted to be large by the nature of the material used. On the other hand, the experiments were repeated and gave essentially the same results. Despite this disadvantage, valuable progress in has been made with actinomycinD Acetabularia cells in the past (Berger et al. 1987; Bonotto, treated 1988; Hammerling, 1934; Harris, 1970; Schweiger et al. 1984). 10 20 30 to days The expression of morphological markers of the species- specific cap of Acetabularia is determined by morphogen- elevated enzyme activity etic substances (morphogenetische Substanzen) originat- ing in the cell nucleus (Hammerling, 1953; Hammerling, translocation of cytoplasm 1963; Werz, 1955). cysts in the caps On the other hand, interspecific transplantation of cell parts provided evidence for the existence of substances Fig. 4. Summary of the nuclear suppression of generative events and their suspension by different means in Acetabularia that are not species-specific. Cap formation, for instance, is acetabulum. Cells were treated as has been described dependent not only upon species-specific morphogenetic (Bannworth and Schweiger, 1983). Exclusion of the cell nucleus substances, but also upon those substances which are not by enucleation, ligation for 5 days or treatment with species-specific (Hammerling, 1953; Werz, 1955). Little is actinomycin D led to an earlier increase in dCMP deaminase known as yet about these nonspecific substances (Ham- activity and to an earlier translocation of chloroplasts from the merling, 1963). stalk into the cap (top bars). In nucleate cells, cysts were also Experiments with Acetabularia acetabulum, nucleate formed earlier. In contrast, the surgery, removal and cells, anucleate cells and cells with homologous nuclear retransplantation of the nucleate basal part into the same cell led to a retarded onset of generative events or had no effect transplants, indicated that the cell nucleus controls (lower bars). generative functions inside the cell that lead to nuclear disintegration, the formation of secondary nuclei and cysts (Bannwarth, 1985a; Bannwarth, 19856; Bannwarth and suspended. In anucleate cells of course, cyst formation was Schweiger, 1983; Bonotto, 1988). In all cases where the cell impossible (Fig. 4). nucleus has been removed or inactivated, an advanced On the other hand, no cytoplasmic translocation and no onset of generative developmental steps has been observed cyst formation has been observed in interspecific grafts (Fig. 4). A suppressor mechanism exerted by the cell (cren1xaceto) (Table 1). Though the caps of these hybrids nucleus has therefore been postulated, and whether this were large and beautiful, obviously disturbances occurred nuclear action is species-specific or not can be answered by which made an ordered progression of differentiation using interspecific grafts. Since the foreign nucleus does processes impossible. It has been reported that cyst not suppress the beginning of the increase of dCMP formation occurs occasionally in crenxacet (acetabulum= deaminase activity as does the homologous nucleus, a mediterranea) hybrids (Beth, 1953; Hammerling, 1953), species-specific nuclear suppression is indicated. but that the cysts were not viable (Werz, 1955). The regulation of visible generative functions such as Since chloroplasts were transported normally into the the translocation of cytoplasm into the cap and cyst cap in anucleate cells, the foreign nucleus in the formation could not be considered in this case, because hybrids must be unable to promote this these processes did not occur in the interspecific hybrids Table 1. Regulation of generative functions in Acetabularia acetabulum and under the influence of a foreign nucleus of Acetabularia crenulata Controls Anucleate Intraspecific grafts Interspecific grafts A. acetabulum A. acetabulum A. acetabulum cre^xaceto Start of the increase normal advanced normal advanced dCMP deaminase activity Translocation of chloroplasts normal advanced normal or retarded not observed from the stalk into the cap Cyst formation normal not observed normal or retarded not observed

866 H. Bannwarth and E. J. de Groot (Table 1). It was therefore important to observe the change regulation and distribution of dCMP deaminase enzyme activity in nucleate and anucleate cells. In Acetabularia 1984 (ed. S. Bonotto, F in enzyme activity at the begin of the generative phase. Cinelli and R. Billiau), pp. 141-161. Belgian Nuclear Center, C.E.N.- The procedure of removal and subsequent retransplan- S.C K., Mol. Belgium. tation of the nucleus-containing basal cell part excluded BANNWARTH, H. (1988). Nucleo-cytoplasmic interactions in cultured the possibility that the early increase in enzyme activity is Acetabularia. In Experimental Phycology, A Laboratory Manual (ed. C due to nonspecific effects of the surgical stress (Bannwarth S. Lobban, D. J. Chapman and B. P. Kremer), pp. 260-271. New York: Cambridge University Press. and Schweiger, 1983). The other possibility that the early BANNWARTH, H., IKEHARA, N. AND SCHWEIGER, H.-G. (1982). increase in enzyme activity is due to the action of the Deoxycytidine monophosphate deaminase in Acetabularia: properties foreign nucleus is also not plausible, since anucleate cells and regulation in the early generative phase. Eur. J. Cell Biol. 27, of Acetabularia acetabulum similarly exhibit this early 200-205. increase and the species-specific action of the foreign BANNWARTH, H. AND SCHWEIGER, H.-G. (1983). The influence of the nucleus on the regulation of the dCMP deaminase in Acetabularia. crenulata nucleus only became apparent 20 days later. Cell Biol. Int. Rep. 7, 859-868. This late increase, characteristic for the species Acetabu- BERGER, S., DE GROOT, E. J., NEUHAUS, G. AND SCHWEIGER, M. (1987) laria crenulata, occurred four days later in the hybrids Acetabularia: a giant single organism with valuable advantages for than it did in the untreated controls (Fig. 3). cell biology. Eur. J. Cell Biol. 44, 349-370. BERGER, S. AND SCHWEIGER, H.-G. (1980). Acetabularia: techniques for The experiments described here clearly exclude non- study of nucleo-cytoplasmic interrelationships. In Handbook of specific substances originating from the nucleus for the Phycological Methods. Developmental and cytological methods (ed. E. regulation described. PolyADP-ribose, for instance, which Gantt), pp. 47-57. Cambridge: University Press. is known to control gene expression and cell differen- BETH, K. (1953). Uber den EinfluB des Kernes auf die Formbildung von Acetabularia in verschiedenen Entwicklungsstadien. Z. Naturf. 8b, tiation (Farzaneh et al. 1982), does not seem to be essential 771-775. in the species-specific control of the succession of generat- BONOTTO, S. (1988). Recent progress in research on Acetabularia and ive steps in Acetabularia. The search for species-specific related . Prog. Phycol. Res 6, 59-235. substances must be focussed on short-lived nucleic acids or BRACHET, J., CHANTRENNE, H. AND VANDERHAEGHE, F. (1955). Recherches sur les interactions biochimiques entre le noyau et le proteins released by the nucleus that control differen- cytoplasme chez les organismes unicellulaires II Acetabularia tiation. mediterranea. Biochim. bwphys. Ada 18, 544-563. The data point to an important additional regulatory DE GROOT, E. J. AND SCHWEIGER, H.-G. (1983). Thymidylate kinase from role of the nucleus in the temporal organization of the cell. Acetabularia: II. regulation during the life cycle. J. Cell Sci. 64, 27-36 The parallels between the dCMP deaminase regulation DE GROOT, E. J. AND SCHWEIGER, H.-G. (1984). Possible translocation of described here and the genetic control of circadian a gene for thymidylate kinase from the chloroplast to the nuclear rhythmicity are noteworthy (Li-Weber et al. 1987). In both genome during evolution. J. Cell Sci. 72, 15—21. cases, the seem to be localized inside the organelles FAKZANEH, F., ZAUN, R., BRILL, D. AND SHALL, S. (1982). DNA strand breaks and ADP-ribosyl transferase activation during cell (chloroplasts, mitochondria), but the nucleus exerts an differentiation. Nature 300, 362-366. influence on the expression of the genes (Schweiger et al. HAMMERLING, J. (1934). Entwicklungsphysiologische und genetische 1984). On the other hand, a DNA sequence, which is Grundlagen der Formbildung bei der Schirmalge Acetabularia. homologous to a short region of the Drosophila period (per) Naturwissenschaften 22, 829-836. locus, may be found at different locations in various HAMMERLING, J. (1953). Nucleo-cytoplasmic relationships in the development of Acetabularia. Int. Rev. Cytol. 2, 475-498. species of (Li-Weber et al. 1989). This locus HAMMERLING, J. (1963). Nucleo—cytoplasmic interactions in plays a fundamental role in the expression of biological Acetabularia and other cells. A. Rev. PI. Physiol. 14, 65-92. rhythms in Drosophila. HAMMERLING, J. AND ZETSCHE, K. (1966). Zeitliche Steuerung der Formbildung von Acetabularia. Umsch. Wiss. Techn. 15, 489-492. Several genes that control early steps in DNA synthesis HARRIS, H. (1970). The expression of genetic information. In Nucleus are also found in the chloroplast genome of some and Cytoplasm (ed. H. Harris), pp. 1-20 Oxford: Clarendon Press. Dasycladacids and in the nuclear genome of others. In LI-WEBER, M., DE GROOT, E. J. AND SCHWEIGER, H.-G (1987). Sequence oerstedii, a phylogenetically old member of the homology to the Drosophila per locus in higher nuclear DNA family, the per locus homologous sequence has been and in Acetabularia chloroplast DNA. Molec. gen. Genet. 209, 1-7. LI-WEBER, M., LEIBLE, M. B. AND SCHWEIGER, M. (1989). Difference in detected in the chloroplast genome, while in Acetabularia the location in Dasycladaceae of a sequence homologous to the cliftonii it has been identified in the nuclear genome (Li- Drosophila per locus. Plant Cell Rep. 8, 169-173. Weber et al. 1989). Similarly, on the basis of inhibitor ROMMELAERE, J. AND HIERNAUX, J. (1975). Model for the positional studies, evidence has been presented that the dTMP differentiation of the cap in Acetabularia. Biosystems 7, 250-258. kinase enzyme is synthesized on the 70S ribosomes in SCHWEIGER, H.-G. (1969). Cell biology of Acetabularia. Curr. Top. Microbiol. Immunol. 50, 1-36. and on 80 S ribosomes in Acetabularia SCHWEIGER, H.-G. (1980). The role of compartmentation in cell mediterranea=acetabulum (de Groot and Schweiger, 1983; morphogenesis and development. In Cell Compartmentation and de Groot and Schweiger, 1984; Schweiger et al. 1986). In Metabolic Channeling (ed. L. Nover, F. Lynen and K. Mothes), pp. both cases, these findings have been interpreted to suggest 455-463. Jena: VEB Gustav Fischer and Amsterdam: Elsevier/North- Holland Biomedical Press. a gene translocation between the chloroplast and the SCHWEIGER, H.-G. (1982). Interrelationship between chloroplasts and the nuclear genome during evolution. nucleo-cytosol compartment in Acetabularia. In Nucleic Acids and Proteins in II. Structure, Biochemistry and Physiology of Nucleic Acids (ed. B. Parthier and D. Boulter), pp. 645-662. We thank Professor W. Dillard for carefully reading the Encyclopedia of Plant Physiology, New Series 14, Part B. Berlin, manuscript, and Mrs C. Fabricius, Mrs L. Neutard and Mrs E. Heidelberg: Springer-Verlag. Schindler for their technical help in preparing it. SCHWEIGER, H.-G., BANNWARTH, H., BERGER, S., DE GROOT, E. J., NEUHAUS, G. AND NEUHAUS-URL, G. (1984). Interactions between compartments in Acetabularia during gene expression. In Compartments in algal cells and their interactions (ed. W. Wiessner, D. Robinson and R. C. Starr), pp. 28-35. Berlin, Heidelberg: Springer- References Verlag. SCHWEIGER, H.-G. AND BERGER, S. (1979). Nucleocytoplasmic BANNWARTH, H. (1985a). Wechselwirkungen zwischen Zellkern und interrelationships in Acetabularia and some other Dasycladaceae. In Zelle in Acetabularia. Unwersitas 40, 153-165. Int. Rev. Cytol. Suppl. 9, pp. 11-44. New York: Academic Press. BANNWARTH, H. (19856). The regulatory role of nucleus and cytoplasm SCHWEIGER, H.-G., DE GROOT, E. J., LEIBLE, M. B. AND TYMMS, M. J. at the early generative phase in Acetabularia mediterranea. (1986). Conservative and variable features of the chloroplast genome

Nuclear control of an enzyme in Acetabularia 867 of Acetabularia. In Regulation of chloroplast differentiation (ed. G. WERZ, G. (1965). Determination and realization of morphogenesis in Akoyunoglou and H. Senger), pp. 467-476. New York: Alan R. Liss. Acetabularia. Brookhaven Symp. Biol. 18, 185-203. SCHWEIGER, H.-G., DEHM, P. AND BERGER, S. (1977). Culture conditions ZETSCHE, K. (1966). Regulation der zeitlichen Aufeinanderfolge von for Acetabularia. In Progress in Acetabularia Research (ed. C. L. F. Differenzierungsvorgangen bei Acetabularia. Z. Naturforsch. 21b, Woodcock), pp. 319-330. New York: Academic Press. 375-379. WERZ, G. (1955). Kemphysiologische Untersuchungen an Acetabularia. Planta 46, 113-153. {Received 24 July 1991 - Accepted 4 September 1991)

868 H. Bannwarth and E. J. de Groot