Journal of the Marine Biological Association of the United Kingdomhttp://journals.cambridge.org/MBI

Additional services for Journal of the Marine Biological Association of the United Kingdom:

Email alerts: Click hereSubscriptions: Click hereCommercial reprints: Click hereTerms of use : Click here

Developmental biology of Acetabularia

Silvano Bonotto

Journal of the Marine Biological Association of the United Kingdom / Volume 74 / Issue 01 / February 1994, pp 93 ­ 106DOI: 10.1017/S0025315400035694, Published online: 11 May 2009

Link to this article: http://journals.cambridge.org/abstract_S0025315400035694

How to cite this article:Silvano Bonotto (1994). Developmental biology of Acetabularia. Journal of the Marine Biological Association of the United Kingdom, 74, pp 93­106 doi:10.1017/S0025315400035694

Request Permissions : Click here

Downloaded from http://journals.cambridge.org/MBI, IP address: 130.194.20.173 on 20 Oct 2012

/. mar. bid. Ass. U.K. (1994), 74,93-106 93Printed in Great Britain

DEVELOPMENTAL BIOLOGY OF ACETABULARIA

SILVANO BONOTTO

Department of Animal Biology, University of Turin, Via Accademia Albertina 17,10123 Torino, Italy

Acetabularia (Dasycladaceae: Chlorophyta) is a giant unicellular marine alga possessinga single nucleus but several millions of chloroplasts and mitochondria. It presents a polargrowth and a peculiar morphological differentiation, comprising the development of abranched rhizoid at its basal end, where the nucleus is located, and the formation ofseveral seriated whorls and then a reproductive cap at the apex of the stalk. Acetabularia isparticularly useful in many fields of cellular and molecular biology. Recent work andcurrent ideas on its developmental biology are summarized and discussed.

INTRODUCTION

Acetabularia (Figure 1) is one of the most famous unicellular algae, reported as amedicinal plant by the Greek physician and pharmacologist Pedianus Dioscorides inthe first century of our era (Bonotto, 1988).

Probably the oldest line drawings of a widely distributed species growing in theMediterranean Sea {Acetabularia acetabulum = A. mediterranea) were published by Mattioli(1586) and by Parkinson (1640). Its 'domestication' in the laboratory, first obtained inGermany with the species A. acetabulum (Hammerling, 1931) and then in Japan with therelated species A. calyculus (Arasaki, 1942), permitted a rapid development of experi-mental work in various fields of cellular and later of molecular biology (Hammerling,1953; Puiseux-Dao, 1970; Berger et al., 1987). Mainly because of its large cell size,Acetabularia is particularly suitable for investigating the subtle relationships which existbetween the nucleus and the cytoplasm, and for studying the intergenomic co-opera-tion inside the eukaryotic cell (Brachet, 1952; Hammerling, 1953; Schweiger, 1969;Schneider et al., 1989). In addition, the obtention of enucleated cells by simply cuttingoff the rhizoid, the preparation of cytoplasts and protoplasts and the isolation ofprimary nuclei, chloroplasts and mitochondria allow refined manipulations and newexperimental approaches to the study of nuclear genes' action on cellular differentia-tion (Berger et al, 1987; Bonotto, 1988).

Particularly useful for investigating the effect of nuclear genes on the morphologicaldifferentiation of the cell are the intra- and inter-specific grafts (Figure 2), first obtainedby Hammerling (1940) between the species A. acetabulum (= A. mediterranea) and A.crenulata, and then widely used for other species (Schweiger et al., 1967; Puiseux-Dao etal., 1970; Bonotto et al., 1971a; the present work). Many new techniques and tools, suchas monolayer spreading in electron microscopy of nucleic acid molecules (Kleinschmidt,

94 S. BONOTTO

DFigure 1. Cap formation in (A) Acetabularia acetabulum (= A. mediterranean (B) A. calyculus; (C,D)Polyphysa peniculus (= A. peniculus). The cell in D, enucleated at the vegetative stage 15 days earlier,has formed two superposed caps, one of them showing some undeveloped rays (arrow). Scale bars-(A) 2-5 mm; (B) 1 mm; (C) 2 mm; (D) 2 mm.

1968), video-microscopy (Trendelenburg et al., 1988), fluorescence microscopy with theaid of DAPI (4'-6-diamidino-2-phenylindole) fluorochrome (Liittke & Bonotto, 1982;Shihira-Ishikawa & Kuroiwa, 1984; De & Berger, 1990) and immunocytochemistry(Menzel, 1986) have been applied to research on Acetabularia. This has considerablyincreased the knowledge of the structural organization of the cell and has allowed thevisualization of the nucleolar rRNA cistrons (Spring et a\., 1974), of the lamp-brushchromosomes with their lateral RNP fibrils (Spring & Franke, 1981), of chloroplast andmitochondrial DNAs (Green & Burton, 1970; Mazza & Casale, 1991), and of the cy-toskeleton components (Menzel, 1986). It was definitely demonstrated that the primarynucleus of A. acetabulum undergoes meiosis with the formation of 20 bivalents, and thekaryotype of this species was established on the basis of the lengths of condensedmetaphase chromosomes (De & Berger, 1990).

The large (up to 150 um) primary nucleus was isolated, injected with exogenousmaterial and reimplanted into enucleated cells (Cairns et al., 1978), allowing the demon-stration of the expression in Acetabularia of heterologous genetic information (seereferences in Bonotto, 1988). Besides, the techniques recently developed by molecular

BIOLOGY OF ACETAB ULARIA 95

Figure 2. Graft of the type ace! pen,. The hybrid cell, obtained by grafting two nucleated basalfragments, one from Acetabularia acetabulum (ace,) and the other from Polyphysa penkulus (pen,), hasformed an 'intermediary' cap. The large number of rays is typical of A. acetabulum, whereas the pear-shaped rays are characteristic of P. penkulus, as those shown in Figure 1C,D. Scale bar: 1 mm.

biologists for research on DNA (restriction enzyme analysis, hybridization, cloning andsequencing) were successfully applied in Acetabularia, opening new perspectives for itsmolecular genetics (Tymms & Schweiger, 1985; Li-Weber et a\., 1987).

Of particular interest, for the understanding of circadian rhythms in Acetabularia(Vanden Driessche, 1967), was the finding that DNA sequences homologous to the per-gene, which is involved in the biological clock expression of Drosophila (Bargiello et al.,1984; Reddy et al, 1984; Zehring et al, 1984), are located in the chloroplast DNA (Li-Weber et al., 1987). This unexpected result, as noted by Berger et al. (1987), is relevant interms of evolution, since Acetabularia is phylogenetically older than the other investi-gated organisms. The above considerations show that, although Acetabularia is a plant,it may be utilized as a model organism for studies in various fields of modern biologicalresearch, including developmental biology.

REPRODUCTIVE MECHANISMS

A number of studies has revealed that Acetabularia, although unicellular, has complexreproductive mechanisms, including both sexual and asexual propagation (Bonotto,1988). Fusion of haploid gametes, originating from different gametangia (operculate

96 S. BONOTTO

Fusion G~©>-,Figure 3. Life cycle of Acetabularia acetabulum. Scheme showing the successive steps leading to cystformation, gamete production and fusion, and zygote settlement, followed by polar growth of thestalk and development of sterile whorls and cap. C, cysts; G, gametes; R, rhizoid containing a residualbody; S, secondary nuclei (haploid); W, whorls; Z, zygote. Cysts of sign + and - produce respectivelygametes + and -. (From Bonotto, 1988.)

Zoospores ( 2n I

( 2n )ZygotesI 2n 1

Figure 4. Scheme showing the different possible types of reproduction of Acetabularia: (A) gametefusion; (B) zoospore germination; (C) cyst germination; (D) gamete parthenogenesis, producing'monster cells'; (E) germination of basal cytoplasm; (F) fusion of secondary haploid nuclei. (FromBonotto, 1988.)

BIOLOGY OF ACETAB ULARIA 97

cysts), with the formation of a diploid zygote, is probably the most important form ofpropagation in nature (Figure 3) and under laboratory conditions. In addition to thismechanism (Figure 4A), five others were reported: zoospore germination (Figure 4B),cyst germination (Figure 4C), gamete parthenogenesis (Figure 4D), germination ofbasal cytoplasm (Figure 4E) and fusion of haploid secondary nuclei in the stalk (Figure4F). Germination of basal cytoplasm, protected in the rhizoid, represents an obviousadvantage for the species, since the cell may survive temporary adverse environmentalconditions and also grazing of the stalk by herbivorous predators such as fish, snailsand amphipods (Bannwarth et ah, 1985). This mode of reproduction from the rhizoidwas also observed in the related genera Bornetella and Halicoryne (Valet, 1968,1969).

Gamete parthenogenesis, first suggested by Hammerling (1934a), was confirmed bymore recent studies (Green, 1976; Primke et ah, 1978; Diibel et ah, 1985). Since gametes ofsign + and - are formed (Figure 3), theoretically it would be possible that haploid plants+ and - are produced if both types of gametes germinate. However, the cells born byparthenogenesis are small and unable to develop normally and form a reproductivecap, and were called by Green (1976) 'monster cells'. Even fusion of haploid secondarynuclei of the same or of opposite sign, observed in A. crenulata, would give rise,respectively, to 'monster cells' or to normal algae (Bannwarth, 1985).

CELL POLARITY AND MORPHOLOGICAL DIFFERENTIATION

An adult Acetabularia cell presents a pronounced polarity, with intracellular morpho-logical and physiological gradients (Hammerling, 1934b; Puiseux-Dao & Dazy, 1970;Issinger et ah, 1971; Schweiger & Berger, 1981). The morphological differentiation,terminating with the development of a cap, may occur even in the absence of thenucleus {i.e. in experimentally enucleated cells) and is accompanied by biochemicalchanges in the various cell compartments (Schweiger, 1980). Most recent work onAcetabularia was done to study the cytological and biochemical bases of polarity andcellular differentiation (Berger et ah, 1987; Berger, 1990a,b; Berger & Kaever, 1992). Apolar organization, called by Berger (1990a) 'primary polarity', is already present in thebiflagellate gamete, in which the location of the nucleus might influence the site oforigin of the flagellar pole. This interpretation is in agreement with the earlier investiga-tions by Crawley (1966,1970), which revealed the ultrastructural polarity of the gamete.This latter showed the single elongated chloroplast, located at the pole opposite to thetwo flagella and bent around the nucleus. It remains unclear whether the mitochondrialsections visible in the gamete micrographs, published by Crawley (1966, 1970) andBerger (1990a), belong to several or only to a single elongated and folded mitochon-drion.

The single chloroplast of the polarized gamete possesses the eyespot apparatus(Crawley, 1966,1970; Kellner & Werz, 1969), which enables the gamete itself to swimtowards the light. On the contrary, the zygote, formed by the fusion of two gametes,swims downwards, away from the light. Consequently, as pointed out by Berger(1990a), the nucleus becomes positioned towards the basal end of the zygote, in which

98 S. BONOTTO

the polarity is preserved. After anchoring of the zygote by the newly formed rhizoid, itsapical pole grows into a stalk, which becomes elongated and develops successivelyseveral sterile whorls of ramified laterals and then a reproductive cap (Berger et al.,1987; Berger & Kaever, 1992). As shown by Gibor (1977) and by Bonotto & Sironval(1977), the direction of stalk growth is influenced by negative geotactic and positivephototactic responses.

The pronounced polarity of an adult vegetative cell may be called 'secondary polar-ity' for distinction from the primary polarity of the gamete. The maintenance of thesecondary polarity is controlled by the nucleus by means of 'morphogenetic substances'(Hammerling, 1934b), which are attributable to long-lived molecules of mRNA(Hammerling, 1963; Brachet, 1968; Kloppstech & Schweiger, 1975, 1976; Berger et al,1987). There are two countercurrent gradients of morphogenetic substances inAcetabularia: those responsible for rhizoid development are more concentrated in thevicinity of the nucleus (baso-apical gradient), whereas those determining stalk, whorlsand cap formation are transported by cytoplasmic streaming and accumulated in theapical region of the cell (apico-basal gradient). It seems, thus, reasonable to assume thata gradient of chemical substances is responsible for the polar nature of morphogenesisin Acetabularia (Schweiger & Berger, 1981; Berger, 1990a). However, the fact that mor-phogenesis in Acetabularia is influenced by numerous factors (Figure 5) suggests that itis a very complex process, which deserves further accurate investigations for a betterunderstanding at the molecular level.

Recent graft experiments between the species A. acetabulum and A. calyculus (Figure6) support the hypothesis that morphogenesis is controlled by species-specific morpho-

LightTemperature Phototropism

WaterMovement

Geotropism

IonTransport Depth

Organic tInorganicCompounds

MORRHOOENESI Salinity

Hormones Time, Season

MetabolicActivity

OrcadianRhythms

Bioelectrical I CytoplasmActivity Morphogenetic Motility

Substances

Figure 5. Diagram showing the main internal (endogenous) and external (exogenous) factors whichinfluence, directly or indirectly, the morphogenesis of Acetabularia. (From Bonotto, 1988.)

BIOLOGY OF ACETABULARIA 99

genetic substances. In fact, in the grafted cell acej calo, which is formed by a basalnucleated fragment of A. acetabulum (ace^ and a long enucleated stalk of A. calyculus(calo), initially the morphogenetic substances accumulated in the apical region of caloquantitatively predominate on those newly produced by the ace, nucleus. Conse-quently, an intermediary cap (IC) is built. However, if this latter is cut off, a typical acecap is formed (Figures 6 & 7). The hypothetical distribution of the morphogeneticsubstances of the type cal and ace is illustrated in Figure 7. It is assumed that the species-specific morphogenetic substances of the type cal progressively disappear (turn-over)and are replaced by those synthesized de novo by the ace nucleus. A graft between twoenucleated fragments, of the type ace0 cal0, might provide further evidence as to thevalidity of the morphogenetic substances hypothesis.

Of great interest is the fact that experimentally enucleated cells are capable of formingone or more whorls and then a cap. Since the nucleus is absent, a differential and

A.acetabulum A.calyculus

Figure 6. Graft of the type ace, cal0. The hybrid cell, obtained by grafting a short nucleated basalfragment from Acetabularia acetabulum (ace^ with a long enucleated one from A. calyculus (cay, formsfirst a typical 'intermediary' cap (IC) and then, after cutting off of IC, a species-specific ace cap.

100

cal

S. BONOTTO

MSm

Figure 7. Schematic representation of the hypothetical distribution of the 'morphogenetic substances'(MS) in Acetabularia calyculus (cal), A. acetabulum (ace) and in the graft ace + cal (ace, cay. In the graftace, cal(, the 'morphogenetic substances' cal (•), accumulated at the tip, turn over and disappear,being replaced by those synthesized de novo by the ace nucleus (O), with the final formation of atypical ace cap.

sequential activation of nuclear genes can be excluded. Consequently, the regulation ofmorphogenesis most probably does not occur at the level of transcription of nucleargenes but at that of translation of stored nuclear mRNAs. In addition, it appears that theenucleated cell must be able to select, from the bulk of stored mRNA, the molecules tobe translated at a given time, and that this ability does not require the presence of thenucleus (Li-Weber & Schweiger, 1985; Berger, 1990b). However, if it seems reasonableto assume, on the basis of biological (enucleation, grafts) and biochemical (extraction

BIOLOGY OF ACETABULARIA 101

and characterization of mRNA molecules) work, that selective translation of storedmRNAs regulates cellular differentiation in Acetabularia, the mechanisms responsiblefor a correct selection and for an exact translation timing of specific mRNAs remain tobe discovered. To add to this complexity is the hypothesis that the perinuclear densebodies, earlier called 'nuclear emissions' (Brachet, 1968; Puiseux-Dao, 1970), which canincrease to as many as 20000 around a fully grown primary nucleus (Spring et al, 1974),might include amplified DNA molecules and perhaps rRNA cistrons, which would betransported into the cytoplasm (Franke et al, 1974; Spring et al, 1974; Berger & Schweiger,1975). It would be worthwhile attempting the isolation of these perinuclear densebodies, which react cytochemically in a way usually considered characteristic of chro-matin, and analysing them biochemically. Moreover, if they really contain DNA mol-ecules, it would be of great interest to know how long these molecules remain in thecytoplasm and, eventually, how they become distributed into the cell.

CONSIDERATIONS ON THE MORPHOGENESIS OF ACETABULARIA

It is known that morphogenesis in Acetabularia is controlled by internal (endogenous)and by external (exogenous) factors (Figure 5). Among these factors, white and bluelight, which are perceived by a photoreceptor probably constituted by a flavin-cyto-chrome-b-protein complex, play an important role (Caubergs et al., 1984; Paques &Brouers, 1984; Schmid, 1984; Vanden Driessche & Caubergs, 1985). As underlined byCastillo et al. (1986), a complex regulation would occur during the time interval betweenthe light photoreception and the morphogenetic response. The mechanisms of suchregulation remain at present unknown in Acetabularia. In addition, the role of hormonesin the control of morphogenesis, a promising topic, was investigated only to a limitedextent (Bonotto, 1988).

Although some particular developmental specific proteins may play a direct role inmorphogenesis (Shoeman & Schweiger, 1982; Shoeman et al., 1983; Berger et al., 1987),the trigger mechanism which induces the transition from the vegetative to the repro-ductive state remain to be discovered. Besides, although it was demonstrated thatcalcium ion concentration affects morphogenesis in Acetabularia (Harrison & Hillier,1985; Harrison et al., 1985; Goodwin & Trainor, 1985), the available information is stillinsufficient to know whether calcium is a 'primary' or a 'secondary' morphogen.

Several authors have presented models for the spatial and temporal regulation ofmorphogenesis in Acetabularia, based on both experimental work and on theoreticalconsiderations (Babloyantz et al., 1975; Rommelaere & Hiernaux, 1975; Puiseux-Dao,1979; Harrison et al, 1981, 1985; Goodwin & Trainor, 1985; Harrison & Hillier, 1985).The two-stage model coceptualized by Harrison et al. (1981, 1985) on the basis of thereaction-diffusion theory, has allowed the visualization, by computer simulation, ofstalk-tip flattening and whorl formation. This approach in the study of Acetabulariamorphogenesis seems particularly promising. However, a strict collaboration betweenexperimental cell biologists and theoretical modellers would be necessary. In fact, someinteresting observations on the developmental biology of Acetabularia remain fre-

102 S. BONOTTO

quently ignored by the modellers. In this context it is of interest to note that Acetabulariamay form, even in the absence of the nucleus, more than one whorl and more than onecap (Figure ID). At the apex of the stalk, whorls (vegetative state) and caps (reproduc-tive state) succeed each other, the former being caducous. Formation of superposedcaps was often observed in A. acetabulum (Bonotto, 1988), more frequently in A. crenulataand sometimes also in Polyphysa peniculus (= A. peniculus). In addition, in the relatedDasycladacean, Halicoryne, up to 30 caps may be formed, always intercalated withwhorls. It appears obvious, from these considerations, that models for the morphogen-esis of Acetabularia should explain not only the serial formation of whorls and of a singlecap at cell maturity, but also the alternating development of whorls and caps, whichmay occur even in the absence of the nucleus (Bonotto et al., 1971b).

As already discussed above, morphogenesis in Acetabularia would be regulated at thetranslational level, the cell having the capability of selecting specific molecules from abulk of stored mRNA (Berger et al., 1987). If a selective translation of mRNA intospecific developmental proteins really occurs, the above reported alternating succes-sion from whorls to caps implies that: (i) the mRNAs responsible respectively for whorland cap formation must be selected alternatively from the bulk of stored mRNA; (ii)after selection and translation, the specific mRNA molecules should not be destroyedby the endogenous RNAse (Schweiger, 1966), but should be kept protected for asuccessive utilization.

CONCLUSION

Research on Acetabularia has progressed substantially in the last 50 years. Neverthe-less, the regulatory mechanisms proposed for polar growth, morphological cell differ-entiation and the alternating formation of whorls and caps remain hypothetical. Directproof that the morphogenetic substances are indeed mRNA molecules is still lacking.Also the real nature of a putative 'primary morphogen' is not yet known with certainty.Finally, the trigger mechanism which determines the transition from the vegetative(stalk and whorls formation) to the reproductive (cap formation) is not yet elucidated.The complexity of the processes involved in Acetabularia morphogenesis would require,for a successful approach, more interdisciplinary work and international collaboration.

Part of this work was supported by the CNR.

REFERENCES

Arasaki, S., 1942. On the life-history of Acetabularia calyculus Quoy & Gaimard. Botanical Magazine,Tokyo, 56, 383-391. [In Japanese.]

Babloyantz, A., Hiernaux, J. & Prigogine, I., 1975. Some remarks on thermodynamics and kineticsof self-organization. In Molecular biology of nudeocytoplasmic relationships (ed. S. Puiseux-Dao),pp. 315-323. Amsterdam: Elsevier.

Bannwarth, H., 1985. On the morphogenetic capacity of secondary nuclei in Acetabularia crenulata.In Acetabularia 1984 BIG 583 (ed. S. Bonotto et al.), pp. 121-138. Mol: Belgian Nuclear Center.

BIOLOGY OF ACETAB ULARIA 103

Bannwarth, H., Schmitz, E., Ropert, H.G. & Timm, U., 1985. Damages on Acetabularia mediterraneacells grown in culture caused by the marine water-flea Elasmopus rapax Costa (Melitidae,Gammaridea, Amphipoda). In Acetabularia 1984, BLG 583 (ed. S. Bonotto et ah), pp. 53-65. Mol:Belgian Nuclear Center.

Bargiello, T.A., Jackson, F.R. & Young, M.W., 1984. Restoration of circadian behavioural rhythmsby gene transfer in Drosophila. Nature, London, 312, 52-54.

Berger, S., 1990a. Acetabularia: a giant unicellular organism for studying polarity. In Experimentalembryology in aquatic plants and animals (ed. H.-J. Marthy), pp. 21-40. New York: Plenum Press.

Berger, S., 1990b. Dasycladaceae: a family of giant unicellular algae ideal for research. In Experimen-tal embryology in aquatic plants and animals (ed. H.-J. Marthy), pp. 3-9. New York: Plenum Press.

Berger, S., Groot, E.J. de, Neuhaus, G. & Schweiger, M, 1987. Acetabularia: a giant single cellorganism with valuable advantages for cell biology. European Journal of Cell Biology, 44, 349-370.

Berger, S. & Kaever, M.J., 1992. Dasycladales. An illustrated monograph of a fascinating algal order.Stuttgart: Georg Thieme Verlag.

Berger, S. & Schweiger, H.G., 1975. The ultrastructure of the nucleocytoplasmic interface inAcetabularia. In Molecular biology of nucleocytoplasmic relationships (ed. S. Puiseux-Dao), pp. 243-250. Amsterdam: Elsevier.

Bonotto, S., 1988. Recent progress in research on Acetabularia and related Dasycladales. In Progressin phycological research, vol. 6 (ed. F.E. Round and D.J. Chapman), pp. 59-235. Bristol: Biopress.

Bonotto, S., Kirchmann, R. & Manil, P., 1971a. Cell engineering in Acetabularia: a graft method forobtaining large cells with two or more reproductive caps. Giornale Botanico Italiano, 105,1-9.

Bonotto, S., Puiseux-Dao, S., Kirchmann, R. & Brachet, J., 1971b. Faits et hypotheses sur le controlede 1' alternance morphogenetique croissance vegetative - differentiation de 1' appareilreproducteur chez les Acetabularia mediterranea Lamouroux, A. crenulata Lamouroux etHalicoryne spicata (Kiitzing) Solms-Laubach. Comptes Rendus de V Academie des Sciences. Paris,272, 392-395.

Bonotto, S. & Sironval, C, 1977. Experimental studies on the phototropism of Acetabulariamediterranea and Acetabularia crenulata. In Progress in Acetabularia research (ed. C.L.F. Wood-cock), pp. 241-247. New York: Academic Press.

Brachet, J., 1952. The role of the nucleus and the cytoplasm in synthesis and morphogenesis.Symposia of the Society for Experimental Biology. Cambridge, 6,173-199.

Brachet, J. 1968. Synthesis of macromolecules and morphogenesis in Acetabularia. In Current topicsin developmental biology, vol. 3 (ed. A. Monroy and A. Moscona), pp. 1-36. New York: AcademicPress.

Cairns, E., Gschwender, H.H., Primke, M., Yamakawa, M., Traub, P. & Schweiger, H.G., 1978.Translation of animal virus in the cytoplasm of a plant cell. Proceedings of the National Academyof Sciences of the United States of America, 75, 5557-5559.

Castillo, F.J., Cotton, G., Kevers, C, Greppin, H., Gaspar, T. & Vanden Driessche, T., 1986.Changes in ascorbic acid content and ascorbate peroxidase activity during the developmentof Acetabularia mediterranea. Differentiation, 33,17-23.

Crawley, J.C.W., 1966. Some observations on the fine structure of the gametes and zygotes ofAcetabularia. Planta, 69,365-376.

Crawley, J.C.W., 1970. The fine structure of the gametes and zygotes of Acetabularia. In Biology ofAcetabularia (ed. J. Brachet and S. Bonotto), pp. 73-83. New York: Academic Press.

Caubergs, R., Vanden Driessche, T. & De Greef, J.A., 1984. A light-inducible cytochrome breduction in the green alga Acetabularia. In Blue light effects in biological systems (ed. H. Senger),pp. 173-176. Berlin: Springer Verlag.

De, D.N. & Berger, S., 1990. Karyology of Acetabularia mediterranea. Protoplasma, 155,19-28.Diibel, S., Neuhaus, G., Berger, S. & Schweiger, H.G., 1985. Characterization of haploid Acetabularia

cells. European Journal of Cell Biology, 38,328-334.Franke, W.W., Berger, S., Falk, H., Spring, H., Scheer, U., Herth, W., Trendelenburg, M.F. &

Schweiger, H.G., 1974. Morphology of the nucleo-cytoplasmic interactions during the devel-opment of Acetabularia cells. 1. The vegetative phase. Protoplasma, 82, 249-282.

104 S. BONOTTO

Gibor, A., 1977. Cell elongation in Acetabularia. In Progress in Acetabularia research (ed. C.L.F.Woodcock), pp. 231-239. New York: Academic Press.

Goodwin, B.C. & Trainor, L.E.H., 1985. Tip and whorl morphogenesis in Acetabularia by calcium-regulated strain fields. Journal of Theoretical Biology, 117,70-106.

Green, B.R., 1976. Abnormal cells resulting from asexual reproduction in Acetabularia(Chlorophyceae, Siphonales). Phycologia, 15,161-164.

Green, B.R. & Burton, H., 1970. Acetabularia chloroplast DNA: electron microscopic visualization.Science, New York, 188,981-982.

Hammerling, ]., 1931. Entwicklung und Formbildungsvermogen von Acetabularia mediterranea. 1.Die normale Entwicklung. Biologisches Zentralblatt, 51,633-647.

Hammerling, J., 1934a. Entwicklungsphysiologische und genetische Grundlagen der Formbildungbei der Schirmalge Acetabularia. Naturwissenschaften. Berlin, 22, 829-836.

Hammerling, J., 1934b. Uber formbildende Substanzen bei Acetabularia mediterranea, ihre raumlicheund zeitliche Verteilung und ihre Herkunft. Archiv fur Entwickungsmechanik der Organismen,131,1-81.

Hammerling, J., 1940. Transplantationsversuche zwischen Acetabularia mediterranea und Acetabulariacrenulata. Note dell Istituto Halo-Germanico di Biologia Marina di Rovigno d' lstria, 2,3-7.

Hammerling, J., 1953. Nucleocytoplasmic relationships in the development of Acetabularia. Inter-national Review of Cytology, 2,475-498.

Hammerling, J., 1963. The role of the nucleus in differentiation in Acetabularia. Symposia of theSociety for Experimental Biology. Cambridge, 17,127-137.

Harrison, L.G. & Hillier, N.A., 1985. Quantitative control of Acetabularia morphogenesis byextracellular calcium: a test of kinetic theory. Journal of Theoretical Biology, 114,177-192.

Harrison, L.G., Hillier, N.A., Glover, J.N.M. & Wallis, P., 1985. Whorl morphogenesis in Acetabulariamediterranea: the role of membrane-bound calcium. In Acetabularia 1984 BLG 583 (ed. S.Bonotto et al.), pp. 109-119. Mol: Belgian Nuclear Center.

Harrison, L.G., Snell, J., Verdi, R., Vogt, D.E., Zeiss, G.D. & Green, B.R., 1981. Hair morphogenesisin Acetabularia mediterranea: temperature-dependent spacing and models of morphogen waves.Protoplasma, 106,211-221.

Issinger, O., Maass, I. & Clauss, H., 1971. Photosyntheseintensitat der Stielregionen von Acetabulariamediterranea. Planta, 101,360-364.

Kellner, G. & Werz, G., 1969. Die Feinstruktur des Augenfleckes bei Acetabularia Gameten undsein Verhalten nach der Gametenfusion. Protoplasma, 67,117-120.

Kleinschmidt, A.K., 1968. Monolayer techniques in electron microscopy of nucleic acid mol-ecules. In Methods in enzymology, vol. 2, part B (ed. L. Grossman and K. Moldave), pp. 361-377.New York: Academic Press.

Kloppstech, K. & Schweiger, H.G., 1975. Polyadenylated RNA from Acetabularia. Differentiation, 4,115-123.

Kloppstech, K. & Schweiger, H.G., 1976. In vitro translation of poly(A)RNA from Acetabularia.Cytobiologie, 13,394-400.

Li-Weber, M., Groot, E.J. de & Schweiger, H.G., 1987. Sequence homology to the Drosophila perlocus in higher plant nuclear DNA and in Acetabularia chloroplast DNA. Molecular GeneralGenetics, 209,1-7.

Li-Weber, M. & Schweiger, H.G., 1985. Evidence for and mechanism of translation control duringcell differentiation in Acetabularia. European Journal of Cell Biology, 38,73-78.

Liittke, A. & Bonotto, S., 1982. Chloroplasts and chloroplast DNA of Acetabularia mediterranea:facts and hypotheses. International Review of Cytology, 77,205-242.

Mattioli, P.A., 1586. De plantis epitome utilissima. Francofurti ad Moenum.Mazza, A. & Casale, A., 1991. Molecular heterogeneities of green algae extranuclear genomes.

Preliminary characterization of two coenocytic green algae extranuclear genomes and phylo-genetic implications. Oebalia, 17, supplement 2, 509-528.

Menzel, D., 1986. Visualization of cytoskeletal changes through the life cycle of Acetabularia.Protoplasma, 134, 30-42.

BIOLOGY OF ACETABULARIA 105

Paques, M. & Brouers, M., 1984. Effect of light on the chloroplast distribution along the stalk ofAcetabularia acetabulum L. (Silva). In Advances in photosynthesis research, vol. 4 (ed. C. Sybesma),pp. 841-844. The Hague: Martinus Nijhoff/Dr W. Yunk Publishers.

Parkinson,J., 1640. Theatrum botanicum (the theatre of plants), pp. 1302-1303. London: Thos Cotes.Primke, M., Berger, S. & Schweiger, H.G., 1978. Protoplasts from Acetabularia: isolation and

fusion. Cytobiologie, 16, 375-380.Puiseux-Dao, S., 1970. Acetabularia and cell biology. London: Logos Press.Puiseux-Dao, S., 1979. Mouvements cytoplasmiques et morphogenese chez 1' Acetabularia

mediterranea. Biologie Cellulaire, 34, 83-90.Puiseux-Dao, S. & Dazy, A.C., 1970. Plastid structure and the evolution of plastids in Acetabularia.

In Biology of Acetabularia (ed. J. Brachet and S. Bonotto), pp. 111-122. New York: AcademicPress.

Puiseux-Dao, S., Valet, G. & Bonotto, S., 1970. Greffes interspecifiques uninuclees, Acetabulariamediterranea et A. peniculus et mobilite des substances morphogenetiques dans le cytoplasme.Comptes Rendus de V Academie des Sciences. Paris, 271,1354-1357.

Reddy, P., Zehring, W.A., Wheeler, D.A., Pirrotta, V., Hadfield, C, Hall, J. & Rosbach, M., 1984.Molecular analysis of the period locus in Drosophila melanogaster and identification of atranscript involved in biological rhythms. Cell, 38, 701-710.

Rommelaere, J. & Hiernaux, J., 1975. Model for the positional differentiation of the cap inAcetabularia. Biosystems, 7, 250-258.

Schmid, R., 1984. Blue light effects on morphogenesis and metabolism in Acetabularia. In Blue lighteffects in biological systems (ed. H. Senger), pp. 419-432. Berlin: Springer Verlag.

Schneider, S.U., Leible, M.B. & Yang, X.-P., 1989. Strong homology between the small subunit ofribulose-l,5-biphosphate carboxylase/oxygenase of two species of Acetabularia and the occur-rence of unusual codon usage. Molecular and General Genetics, 218, 445-452.

Schweiger, H.G., 1966. Ribonuclease Aktivitat in Acetabularia. Planta, 68,247-255.Schweiger, H.G., 1969. Cell biology of Acetabularia. Current Topics in Microbiology and Immunology,

50,1-36.Schweiger, H.G., 1980. The role of compartmentation in cell morphogenesis and development. In

Cell compartmentation and metabolic channeling (ed. L. Nover et al.), pp. 455-463. Jena: VEBGustav Fisher and Amsterdam: Elsevier/North-Holland Biomedical Press.

Schweiger, H.G. & Berger, S., 1981. Pattern formation in Acetabularia. In Cytomorphogenesis inplants. Cell biology monographs, vol. 8 (ed. O. Kiermayer), pp. 119-145. Wien: Springer Verlag.

Schweiger, H.G., Master, R.W.P. & Werz, G., 1967. Nuclear control of a cytoplasmic enzyme inAcetabularia. Nature, London, 216, 554-557.

Shihira-Ishikawa, I. & Kuroiwa, T., 1984. Morphological transition of the nucleus during thewhole life cycle of Acetabularia calyculus Quoy & Gaimard. Japanese Journal of Phycology, 32,147-157.

Shoeman, R.L., Neuhaus, G. & Schweiger, H.G., 1983. Gene expression in Acetabularia. III.Comparison of stained cytosolic proteins and in vivo and in vitro translation products. Journalof Cell Science, 60,1-12.

Shoeman, R.L. & Schweiger, H.G., 1982. Gene expression in Acetabularia. II. Analysis of in vitrotranslation products. Journal of Cell Science, 58,35-48.

Spring, H. & Franke, W.W., 1981. Transcriptionally active chromatin loops of lamp-brush chro-mosomes at physiological salt concentrations as revealed by electron microscopy of sections.European Journal of Cell Biology, 24, 298-308.

Spring, H., Trendelenburg, M.F., Sheer, U., Franke, W.W. & Herth, W., 1974. Structural andbiochemical studies of the primary nucleus of two algal species, Acetabularia mediterranea andAcetabularia major. Cytobiologie, 10,1-65.

Trendelenburg, M.F., Meissner, B., Troster, H., Berger, S. & Spring, H., 1988. Direct visualizationof intranuclear lamp-brush chromosome gene domains using videomicroscopy. Cell BiologyInternational Reports, 12,737-763.

Tymms, M.J. & Schweiger, H.G., 1985. Tandemly repeated non-ribosomal DNA sequences in thechloroplast genome of an Acetabularia mediterranea strain. Proceedings of the National Academy ofSciences of the United States of America, 82,1706-1710.

106 S. BONOTTO

Valet, G., 1968. Contribution a V etude des Dasydadales. Thesis, University of Paris.Valet, G., 1969. Contribution a 1' etude des Dasycladales. 2. Cytologie et reproduction. 3. Revision

systematique. Nova Hedwigia, 17,551-644.Vanden Driessche, T., 1967. The nuclear control of the chloroplasts circadian rhythms. Science

Progress. London, 55, 293-303.Vanden Driessche, T. & Caubergs, R., 1985. Inhibiting the transduction of blue light signals in

Acetabularia. In Acetabularia 1984 BLG 583 (ed. S. Bonotto et al.), pp. 91-100. Mol: BelgianNuclear Center.

Zehring, W.A., Wheeler, D.A., Reddy, P., Konopka, }., Kyriacou, C.P., Rosbach, M. & Hall, J.C.,1984. P-element transformation with period locus DNA restores rhythmicity to mutant,arrhythmic Drosophila melanogaster. Cell, 39, 369-376.