third symposium onacetabularia

Protoplasma 83, 167--183 (1975)

by Springer-Verlag 1975

T h i r d Sympo s iu m on Acetabularia

Organized by

S. Puiseux-Dao

Paris, July 11-12, 1974 Laboratoire de Biologie Cellulaire Vdg&ale Universitd de Paris VII

168 Third Symposium on Acetabularia

Messenger Ribonucleoproteins of Acetabularia

A. B. ALEXEEV, S. L. STVOLINSKY, L. A. TIKHOMIROVA, A. A. YAZYKOV,

a n d T. N . ZU~AREV

Institute of Genetics and Selection of Industrial Microorganisms Moscow, USSR

It has been shown previously that in regenerating cells of A. mediterranea ribonucleoprotein particles occur with sedimentation coefficients of 20 S, 40 S and 80-100 S and a buoyant density of 1.4 g- cm - z [1]. The present work reports on further investigations of the above mentioned particles. Separate centrifugation of nuclei and cytoplasm from regenerating cells was carried out. RNP particles as characterized above were found both in nuclei and in cytoplasm. This was revealed by experiments with pulse (30 minutes) and continuous (16 hours) H3-uridine labeling. In the pulse label experiments an increase in the activity was observed in the zone corresponding to the 80-120 S RNP particles. In experiments with pulse and 16 hours label 9 S and 12 S RNA are found in the nucleus. In pulse label experiments not only precursors of ribosomal RNA are found in the nucleus but also RNA with the sedimentation coefficients exceeding 50 S, which seem to be presursors of m-RNA. In the 20 S and 40 S RNP particles 9 S and 12 S RNA, respectively, were found. Electron microscopic investigation revealed that the 40 S fraction contained particles with the size of about 200 A. Material of the sucrose gradient fraction containing the 40 S RNP particles was injected into non-growing anucleate fragments of A. crenulata. In about 10% of the cases the beginning of the morphogenesis process was induced at one of the fragment ends. No morphogenesis was observed after injection of the preheated 40 S RNP particle fraction or the ribosome fraction. We suggest that the material isolated from regenerating cells of Acetabularia might be identical with the "morphogenetic substances" of Acetabularia postulated by HKMMER- LiNG [2]. May be, they should be termed the H~.mmerling particles or, briefly, the H-particles. Then such particles having different sedimentation constants should be denoted as I-I-20, H-40, H-100 particles.

[1] ALEXEEV, A. B., M. I. BETINA, S. L. STVOLINSKY, A. A. YAZYKOV, and T. N. ZUBARrV, 1974: Plant Science Letters 3, 297.

[2] H.~.MMERLING, J., 1963: Ann. Rev. Plant Physiol. 14, 65.

Fractionation and Characterization o f the Chloroplast Membranes of Acetabular ia med i t e r ranea

K. APEL

Harvard University, Biological Laboratories, Cambridge, MA 02138, U.S.A.

The chloroplast membranes of A. mediterranea were fractionated in the presence of Triton X 100 and the subfractions were separated on sucrose gradients. Two fractions were obtained which have been so far characterized by their chlorophyll content, their fluorescence properties, their structure and their protein composition. There is strong

Third Symposium on Acetabularia 169

evidence that the two fractions are enriched in PS I or PS II, respectively. Two chlorophyll- protein complexes of 120,000 and 67,000 dalton were isolated and characterized. The 67,000 dalton chlorophyll-binding protein is part of photosystem II and is composed of two subunits of 23,000 and one subunit of 21,000 dalton.

Activities of a Mannose-Polymeriz ing Enzyme and GDP-Mannose Pyrophosphorylase during the Morphogenesis of Acetabular ia med i t e r ranea

PETER BACHMANN a n d KLAUS ZETSCHE

Botanisches Institut der Universitiit, D-6300 Giessen, Federal Republic of Germany

The cell wall polysaccharides of Acetabularia mainly consist of mannose. Interesting questions are: The temporal and spatial activities of the GDP-mannose pyrophosphorylase and the mannose-polymerizing enzyme during morphogenesis, and the correlation between the activities of these two enzymes. To answer these questions experiments were carried out with 14C-sodium bicarbonat and labelled GDPM. We tested five morphological stages (from whorl stage to cyst-cap stage) and apical, middle and basal anucleate fragments of the 2nd stage (length 22 ram, cap diameter 3 ram). The experiments with whole plants reveal a temporal correlation between 14CO~-fixation into D-mannose of the cell wall and cap formation. Doing ~4CO2-fixation with segments 73~ of the labelled D-mannose is incorporated into the cell wall of the apical fragment. GDP-mannose pyrophosphorylase activity of whole algae shows a continuous decrease from stage 1 to 5, and from basal to apical. The mannose-polymerizing enzyme demonstrates high activity from stage 1 to 4 and low activity in the 5th stage. The polar distribution of the enzyme shows a remarkable gradient with its maximum value in the apical segment. The 14CO~ experiments and the data on the GDP-mannose pyrophosphorylase indicate, that this enzyme cannot be a dominating regulator for the mannan synthesis. On the other hand the activity of the mannose-polymerizing enzyme corresponds quite well to the 14CO2 incorporation. One may conclude that the mannose polymerizing enzyme is a dominating factor in cell wall formation,

Some Characteristics of the Thymidine Phosphorylating Enzyme (TPE) in Acetabular ia

HORST BANNWARTH

Max-Planck-Institut fiir Zellbiologie, D-2940 Wilhelmshaven, Federal Republic of Germany

Thymidine phosphorylation dramatically increases when a maximum diameter cap is formed in nucleate as well as in anucleate Acetabularia cells. In vivo experiments have indicated that this regulation is due to a change either in thymidine uptake or in the conversion of thymidine into thymidine monophosphates.


Using deoxyribose labelled 3H-thymidine as a substrate it was shown that the site of regulation is a thymidine kinase. Interest was thus focused on the characteristics of this enzyme. At 25 ~ the phosphorylating reaction proceeds linearly for at least 120 minutes. The temperature optimum is 40 ~ and the pH-optimum 7.4. In the crude extract, the half life of the enzyme is about 6 hours at 0 ~ At an ATP concentration of 8 mM the K m for thymidine was found to be 7 • 10 -6 M. A feed-back inhibition was not observed over the range from 0.02 mM to 0.4 mM TTP. After a 20 minutes centrifugation at 20,000 • g 800/0 of the enzyme activity is localized in the sediment.

Effects of Transcription and Translation Inhibitors on the Regulation of the Thymidine Phosphorylating Enzyme (TPE) in Acetabular ia

HORST ]~ANNWARTH

Max-Planck-Institut fiJr Zellbiologie, D-2940 Wilhelmshaven, Federal Republic of Germany

An increase in thymidine kinase activity has been observed in Acetabularia at the stage when the caps attain their maximum size. A similar regulation takes place in cells enucleated two weeks prior to cap formation. Inhibitors of transcription and of translation were added 3 days prior to the onset of the increase in enzyme activity. Puromycin (30rag/l) and chloramphenicol (100mg/1 and 10 rag/l) inhibited the increase in activity almost completely. The addition of cycloheximide (0.1 mg/1) did not affect the increase, but the drug had a stimulatory effect at higher concentration (2 rag/l). The transcription inhibitors actinomycin D, ethidium bromide, and rifampicin had different effects. Actinomycin D (10 mg/1) hardly affected the increase in enzymic activity, Ethidium bromide (25 rag/l) and particularly rifampicin (20 rag/l) were observed to be inhibitory. One may conclude that the thymidine klnase is translated on 70 S ribosomes.

Formation and Ultrastructure of Secondary Nuclei in the Rhizoid of Acetabular ia med i t e r ranea

SIGRID BERGER

Max-Planck-Institut fiJr Zellbiologie, D-2940 Wilhelmshaven, Federal Republic of Germany

Secondary nuclei of Acetabularia mediterranea were observed in the same rhizoid with the primary nucleus, in rhizoids containing only secondary nuclei and during migration through the stalk. At the time of initiation of the generative phase the primary nucleus and the surrounding cytoplasm undergo distinct fine structural changes. After completing this phase, the first secondary nuclei can be detected in the perinuclear region. These secondary nuclei have no nueleoli. They apparently undergo divisions since mitotic figures can be detected. Once many secondary nuclei have formed, they begin to migrate in clusters via the stalk to the cap rays.


Ultrastructure of Nuclei After Implantation

SIGRID BERGER

Max-Planck-Institut ftir Zellbiologie, D-2940 Wilhelmshaven, Federal Republic of Germany

The primary nucleus of Acetabularia rnediterranea undergoes morphological changes during the vegetative phase of the life cycle. These differences make it possible to distinguish between "young" and "old" nuclei by means of electron microscopical techniques. Such "young" and "old" nuclei were isolated and implanted into anucleate cells of different ages; "young" nuclei were implanted into "old" cytoplasm and vice versa. It could be shown that the cytoplasm effects a change in the morphology of the cell nucleus. The morphology of old nuclei in young cytoplasm is changed to the morphology of young nuclei, while young nuclei in old cytoplasm assume the features of old nuclei after a short time.

Acetabularia haemmerlingi, a New Species

SIGRID BERGER


A new species of the family Dasycladaceae was found near the British Solomon Islands. The structure of this species was studied by use of light and scanning electron microscopy. The characteristics of this cell, namely, number of cap rays, number of protuberances per segment of corona superior, shape of cap rays and shape of corona superior and corona inferior segments were compared with the characteristics of the known species of Dasycladaceae. The new species has been classified as a member of the subfamily Acetabularioideae, genus Acetabularia and section Acetabuloides. It has been named Acetabularia haemmerlingi.

On the Mechanism of Activation of Glyceraldehyde 3-Phosphate Dehydrogenase of Acetabularia

ULRIKE BOEGE, FRANK BOEGE, and KLAUS ZETSCHE

Botanisches Institut der Universit~it, D-6300 Giessen, Federal Republic of Germany

In fully developed plants there are two forms of NADP-dependent glyceraldehyde 3-phosphate dehydrogenase activity: irradiated plants show much higher activity than plants which have been stored in the dark. The process of light activation takes about ten minutes in vivo; it can be replaced by incubation of the dark enzyme extract with NADPH, The suggestion of ZIrGLrR et al., that NADPH acts as a positive allosteric effector~ cannot be accepted at least for Acetabularia for the following reasons: There is no decrease in the activity of the light enzyme after ammonium sulphate fractionation or gel filtration on Sephadex. Moreover, the extract of dark plants regains the full


light activity without addition of any effector if it is stored in the dark for several hours at low temperature. This "self-activation" can be accelerated by dialysis. In the same way full light activity can be regained by treatment of the dark enzyme by gei filtration within half an hour. The ability to activate the enzyme by incubation with NADPH is lost during ammonium sulphate fractionation. These results lead to the hypothesis that there must be an inhibitor, which is slowly destroyed in the extract of dark plants, which is rapidly destroyed in the presence of NADPH, and which can be withdrawn by dialysis, gel filtration or ammonium sulphate fractionation. This inhibitor is thought to act directly on the enzyme. The light activation would then be a process of destruction of the inhibitor, in which NADPH participates.

E n e r g y C o n s u m p t i o n o f t h e E l e c t r o g e n i c P u m p

i n A c e t a b u l a r i a m e d i t e r r a n e a

D. GRADMANN a n d G. BOKELOH

Institut fiir Biologie I der UniversitS.t, D-7400 Tiibingen, Federal Republic of Germany

The total ATP consumption of Acetabularia cells has been determined by measuring the initial decrease of ATP upon sudden interruption of phosphorylation by the uncoupler CCCP. Based on the analog circuit of the membrane, with two passive diffusion channels (K+ and C1--) and an electrogenic pump (C1--) in parallel, the power of the electrogenic pump (in the range of 10~W/cm e) has been determined by flux, voltage and conductance measurements under various conditions. It turned out to be in the same order of magnitude as the total ATP supply.

P l a s t i d D N A i n A c e t a b u l a r i a m e d i t e r r a n e a

S. BONOTTO, P. LURQUIN, W. BAtYeNS, P. CI-IARL~S,

D. HOURSIANGou-NEUBRUN, A. MAZZA, G. TRAMONTANO, a n d B. FeLLUGA

D@artement de Radiobiologie, C.E.N./S.C.K., Mol, Belgium, Laboratoire de Biologie Cellulaire V~g&ale, Universitd Paris VII, France, and Laboratorio Internazionale di Genetica e Biofisica, Napoli, Italy

Though Acetabularia was extensively studied to determine some of the interelations between the nucleus and the chloroplasts, their plastid DNA is still not well known. So some investigations were made to give more informations on this DNA. Acetabularia chloroplasts were extensively purified by differential centrifugation and their purity was checked by electron microscopy. Although no bacterial contaminants were found, some mitochondria remained stuck to the plastidal membrane. Plastid DNA was prepared by a method involving repeated treatments by pronase and ribonuclease or by molecular sieving on agarose, performed at high ionic strength. Ultracentrifugation on CsC1 gradient, performed with both the preparative and the analytical centrifuge, has shown that chloroplast DNA has a buoyant density in the range of 1.704, corresponding to 44~ G + C. The


length of the DNA molecules was determined by the technique of ][,ANG and MITANI. It was found that the DNA prepared by molecular sieving contains linear molecules having a size up to 16 ~tm. But larger molecules (up to 200 ~t) were measured. These findings suggest that A. mediterranea chloroplast DNA probably consist of relatively long molecules, which are broken during preparation.

Changes of pH in Culture Solution of Acetabular ia Caused by Proton Flux

ERNST P. O. BR)/NDLE, RENATE K6TTER, and KLAUS ZETSCHE

Botanisches Institut der UniversitY/t, D-6300 Giessen, Federal Republic of Germany

Proton dislocation results in a pH-change in the culture solution of Acetabularia mediterranea. This dislocation of protons is linked to photosynthesis. The proton flux proceeds through the chloroplast membrane and extends across the plasmalemma. Evidence for these assumptions comes from labelling experiments with 14CO~ and experimental alterations of the external HCO~---concentrations with N~, HC1 or NaOH. Therefore we could exclude the possibility that the pH-change in the culture solution is due to organic acids or HCQ---ions. Diamox, an inhibitor of the carboanhydrase, also inhibits the 14CO_~-fixation by 90~ whereas the proton flux is inhibited only by 30~ Furthermore, an increase of the proton flux by about 160~ by DNP does not increase the 14CO~-fixation. Under continuous light conditions the pH of the medium exhibits a rhythm with a period length of 23 hours. This rhythm is similar to other circadian rhythms in Acetabularia mediterranea as those, e.g., for CO~-fixation and O2-production. Compared to the CO 2- fixation and the O2-production, the pH-oscillation is phase-shifted, The proton flux of isolated chloroplasts is quantitatively comparable to that of intact cells. Experiments with inhibitors of cyclic and non-cyclic electron flow as CCCP and DCMU indicate that pH-changes in the culture solution and the non-cyclic electron flow in the chloroplasts are closely connected with each other. It is suggested that correlations exist between proton flux and calcification as well as the polarity of the Acetabularia cell.

RNA Transport from the Nucleus to the Cytoplasm

A. C. DAZu H. BORGm, J. GAUCHERY, and S. PuIsEux-DAO

Laboratoire de Biologie Cellulaire V~g&ale, Universit~ Paris VII, France

Acetabularia mediterranea may provide a model system for an in vivo study of RNA transport from the nucleus to the apical part of the cell where growth occurs. In order to study the kinetics of this transport and the effect of light, pulse-chase experiments were performed under various conditions (light: dark 12-12; continuous light; continuous dark) using normal and regenerating algae. The migration of the non chloro- plastic TCA insoluble material was followed in basal and apical fragments.


In the dark there is no nucleo-cytoplasmic RNA transport if algae have been cultivated without light during 2 weeks although the nuclei incorporate '~H-uridine as can be observed autoradiographically. In the light transport of labelled RNAs to the basal and apical parts of the cell is very rapid. The migration pattern seems to vary with the stage of development of the Acetabularia. In the case of algae sectionned 1 cm above the rhizoids just after the incubation with 8H-uridine, results are the most reproducible; two phases of RNA migration are observed.

Fatty Acids Biosynthes i s in Acetabular ia med i t e r ranea

j . P. DUBACQ a n d D. HOURSIANGOU-NEUBRUN

Laboratoire de Physiologie Cellulaire, 12 Rue Cuvier, 75005 Paris, France, Laboratoire de Biologie Cellulaire V6g&ale, Universit6 Paris VII, France

Biosynthesis of fatty acids has been followed by 114C acetate incorporation in vivo and in isolated chloroplasts. Palmitate and oleate are the two major products of acetate incorporation in lipids, as well by whole cells as by plastids. The polyunsaturated fatty acids biosynthesis is very slow in vivo and does not appear in vitro in the plastidal fraction. The fatty acid labelling is different in each lipid class. During the formation of the cap the fatty acids metabolism is more important in vivo and in vitro. In the dark acetate incorporation is lower and no polyunsaturated fatty acids synthesis could be noticed. Returning to light after dark treatment enhances the fatty acids metabolism.

The Nuclear Envelope and the Perinuclear Lacunar Labyr inthum

W. W. FRANKZ, S. Ber, GrR, U. SCrtZER, a n d M. F. Tr, rNI)ELZNBtSRG

Deutsches Krebsforschungszentrum Heidelberg, and Max-Planck-Institut fiir Zellhiologie, D-2940 Wilhelmshaven, Federal Republic of Germany

From the ultrastructural studies of the nuclear envelope of the primary nucleus and associated cell components a scheme is derived in which emphasis is laid on the development of a special perinuclear membranous apparatus, primarily by a cisterna of an extended vacuolar labyrinthum. This cisterna closely surrounds and parallels the nuclear envelope in a distance of ca. 100 nm and thus gives rise to the formation of an "intermediate zone" between the nuclear envelope and this accessory cisterna. This intermediate zone is a zone of exclusion for most of the organelles and particles characteristic of both the cytoplasm and the nuclear interior. The "secondary" perinuclear cisterna contains fenestrae (pores) which, however, are different in ultrastructure from the typical pore complexes of the nuclear envelope and annulate lamellae. This organization seems to be unique among the eukaryotic cells, with the only reported exception in some marine ciliates. The average nuclear pore flow rate of


ribosomal RNA equals to 0.3 ribosome equivalents/pore/rain. It is obvious that it is not the nuclear pore complexes but the fenestrae in the lacunar labyrinthum cisterna ensheathing the nucleus which dimensionally limit the nucleocytoplasmic exchange.

3 6 C h l o r i d e F l u x e s o f Acetabular ia

RAINER M. GLASEL a n d KI~AUS ZETSCHE

Botanisches Institut der Universit~t, D-6300 Giessen, Federal Republic of Germany

In Acetabularia mediterranea ionic relation have been analysed under different conditions. Before testing possible differences of the chloride efflux of the polar Acetabularia cell, we established efflux-kinetics of whole plants by using ~oC1 in artificial seawater. The wash-out kinetics of our cells show a typical biphasic shape--in contrast to other findings, which describe the washout as a single kinetic compartment. Mathematical analysis of these curves gave evidence for at least two superimposed exponential functions, which can be separated by extrapolation and substraction. Following a model of two intracellular compartments in series (described by PIT~AN 1963, CRAM 1968, PIERCE and HIGIN-BOTHAM 1970) we tried to apply a compartment-analysis, in order to show that the slower phase of washout reflects the efflux from the vacuole while the fast one is due to the efflux out of the cytoplasm. If our system fits the model of single compartments in series--and this interpretation holds only if the cells have large vacuoles and if ion efflux out of the vacuole proceeds mainly via the cytoplasm--it would be possible both to calculate ion concentrations of each compartment and to calculate the one-way fluxes at the tonoplast and plasmalemma membrane. When doing so we enfaced some problems: flux rates calculated in this manner are rather high compared with other published values and especially under dark conditions we some- times observed fluxes which were not in equilibrium as should be the case under steady- state conditions. We hope that some more experiments, including influx measurements will give more evidence and allow the method to be applied to morphogenetic questions.

Cellulose i n Acetabular ia C y s t W a l l s

WERNER HERTH

Lehrstuhl for Zellenlehre, Universifilt Heidelberg, Federal Republic of Germany

The alkali-resistant material from the cyst walls of the green alga Acetabularia mediterranea was investigated by X-ray diffraction and electron microscopy and by hydrolysis and thin layer chromatography. The predominant structural polysaccharide is cellulose I which occurs in ribbon-shaped fibrils of high crystallinity. Since in other parts of this plant the exclusive structural polysaccharide is a f3-1,4-mannan this result demonstrates a strict change in the chemical nature of the structural cell wall polysaccharide in two phases of the life cycle.

176 Third Symposium on AeetabuIarla

Ultrastructure of Lamellae from Acetabularia mediterranea Plast ids

D. HOURSIANGOU-NEUBRUN, M. GUILLO, and S. I)UISEuX-DAo

Laboratoire de Biologie Cellulaire V6g&ale, Universit6 Paris VII, France

Plastid lamellae were obtained from isolated plastids by osmotic shock. They were examined with the electron microscope using the negative staining technique. Several types of structures have been observed. They confirm that differences exist between apical and basal plastids and that the morphology of the lamellae depends on experimental conditions (culture medium, darkness, enuclation). These observations give informations on thylacoid formation and degradation and on structural interrelations between lamellae in different parts of the cell under light or dark conditions.

Phase Shift ing the Photosynthet ic Rhythm in Anucleate Acetabularia with Periods of Darkness

MARLENE W. KARAKASHIAN

Max-Planck-Institut far Zellbiologie, D-2940 Wilhelmshaven, Federal Republic of Germany

The circadian rhythm of photosynthetic oxygen evolution in Acetabularia persists for weeks in individual anucleate cells maintained under constant conditions of temperature and illumination (MER~ENHAGEN and SCI-IWrIG~R 1973). Since the circadian mechanism in anucleate cells is also capable of entraining to a new light-dark cycle (SwEENEY and HAxo 1961), it was of interest to examine the effects of non-repeated perturbations of the constant light regimen of such cells. A series of experiments was undertaken in which periods of darkness were introduced at different times in the circadian cycle. These studies reveal a differential sensitivity to dark interruptions at different times of day. The phase response profile generated by such dark treatments is the reciprocal of the profiles generated in other systems using bright light treatments. These findings support the conclusion that the cytoplasm contains all the components which are necessary for the normal expression and function of the circadian clock mechanism in Acetabularia.

C y t o s o l - R i b o s o m e s in Acetabularia: Distr ibut ion and Migration Within the Cell

KLAUS KLOPPSTECH


Prior to answering basic questions related to translational control of gene expression in Acetabularia, it is necessary to know more about ribosome distribution within the cell and their rate and mode of migration. Recent studies revealed that 80 S ribosomes are almost evenly distributed throughout the cell. Ribosome migration was followed by means of


pulsechase experiments. Newly formed 80 S ribosomes appear as free ribosomes, two thirds of which are integrated into polysomes. The newly-formed ribosomes are almost entirely restricted to the vicinity of the nucleus and are integrated into membrane structures within 5 days. Labelled ribosomes can be detected in the apical half of the cell only after a period of about 10 days. Again, the greater part of the apical ribosomes is membrane bound. Three weeks later, labelled ribosomes are largely confined within the apical half of the cell. Since the cell exhibits apical growth the ribosomes must have reached the apex by a direct transporting and not a random mechanism. The rate of migration can be estimated to be approximately 2 mm per day. The half life of the ribosomes is roughly two months. The newly formed ribosomes possess a homogeneous density of 1.52 g/ccm. After three weeks, the particles exhibit densities between 1.50 and 1.56 g/ccm. Whether this heterogeneity indicates different functional states of the ribosomes or integration into different structures cannot be decided at the moment.

P o l y - A C o n t a i n i n g R N A i n A c e t a b u l a r i a

KLAUS KLOPPSTECH

Max-Plan&-Institut fiir Zellbiologie, D-2940 Wilhelmshaven, Federal Republic of Germany

The existence of a poly-A chain at the 3' end in most RNA species is useful for the detection and isolation of mRNA. It has been shown that non-labelled RNA hybridizable with aH-poly-U exists in all regions of the Acetabularia cell, but is present in somewhat higher concentration in the basal section. Cells of A. mediterranea 15 mm in length contain 0.7 ng poly-A per cell, those 25 mm in length 1.7 ng poly-A per cell. Thus, in nucleated cells the amount of poly-A increases by two-fold within two weeks, whereas in the enucleated cell this amount decreases by about 30~ in the same time period. These experiments do not give any insight into the time and place of synthesis of the poly-A carrying RNA. Regenerating cells appear suitable objects for the investigation of the de novo synthesis of poly-A containing RNA, since after removing 900/0 of the cytoplasm, a de novo synthesis of informational RNA is to be expected. Cordycepin (50 vg/ml), known to inhibit polyadenylation within the nucleus, almost completely inhibits the growth of the rhizoids in regenerating cells, indicating that some RNA cannot be transported to the cytoplasm. That de novo mRNA synthesis is occurring, is supported by the observation that regenerating cells show at least a two-fold increase in the amount of poly-A bearing RNA present within 3 days after amputation. The increased rate is maintained for a period of at least 8 days and can be inhibited by more than 70o/0 by cordycepin.

Inf luence o f L i g h t o n the G e r m i n a t i o n o f C y s t s

o f A c e t a b u l a r i a m e d i t e r r a n e a

HANS-ULRICH K o o P

Institut ftir Pflanzenphysiologie und Zellbiologie, Freie UniversitY/t, D-1000 Berlin 33

A technique for the growth of uniformly reacting populations of cysts of Acetabularia mediterranea and for quantitative measurement of cyst germination are developed. Cysts of A. mediterranea can be induced to germinate by exposure to the atmosphere.

Protoplasma 83/1--2 12


Germination rates are very low in young cysts. They increase during exposure to total darkness. This "maturation of cysts" is found to be completed after a period of 12-15 weeks. Germination rates of cysts that have passed the maturation period exceed 90% in continuous white light and 80% in darkness. Cysts germinate in less than two days in darkness and less than four days in light. The influence of temperature at a range of 15 ~ to 25 ~ on germination kinetics is studied in light and darkness. Germination is accelerated with increasing temperature up to 21 ~ At higher temperatures germination is delayed in light but the time of germination remains constant in darkness. Rates of germination are not altered by the influence of temperature in light while in darkness there is a dramatic decrease at temperatures higher than 21 ~ From these findings it is concluded that cyst germination in Acetabularia rnediterranea does not need any light but is influenced by light dependent systems. The influence of light is strongest at elevated temparatures.

A M e t a b o l i c In f luence on 125I-Insulin F ix a t io n to Acetabular ia med i t e r ranea

F. LEGr, os , I. DUMON, B. HANSON, J . JEANMART, a n d V. CONARD

Laboratoire de Physiopathologie, B-1000 Bruxelles, Belgique

Kinetics of 1"2q-insulin fixation to Acetabularia, competition between radioactive and native hormones for cellular binding, acceleration of radioactive insulin release in the presence of native hormone, effect of various concentrations of trypsin, phospholipase and con- canavalin A on cell labeling agree with the results obtained with animal cells or isolated cell membranes indicating a specific binding of insulin to glycoprotein receptor sites. The number of membrane receptors increases when cells are incubated in darkness for 0 to 15 days.

S u s t a i n e d Current P u l s e s and T r a n s c e l l u l a r Current d u r i n g the R e g e n e r a t i o n of Acetabularia medite , 'ranea

B. NOVAK

Biocentrum, Basel, Schweiz

A transcellular current was measured in the enucleated posterior stalk segments (EPSS) of Acetabularia mediterranea. An inhibition of regeneration was observed when the current was prevented to flow through the EPSS. The regeneration of EPSS was correlated with the occurrence of the transeellular current. Sustained current pulses superimposed the steady state transcellular current. The current pulses originate always at the regenerating end of EPSS. It is suggested that the transcellular current and the current pulses cause the polar transport of the morphogenetic substances towards the growing tip, and therefore they represent the beginning of the regeneration processes.

Prote in Kinase of Acetabularia

Third Symposium on Acetabu&ria 179

H . S. PAI, D . DEHM 1, M. SCHWEIGER, H . J. RAHMSDORF, H . I)ONTA,

M. HIRSCH-KAUFFMANN, a n d H . G. SGH\VEIGER 1

Max-Planck-Institut fiir Molekulare Genetik, Berlin-Dahlem, and 1 Max-Planck-Institut fiir Zellbiologie, Wilhelmshaven, Federal Republic of Germany

Acetabularia major and A. mediterranea as well as 16 other Dasycladaceae contain protein kinase activity which transfers phosphate from ATP to serine or threonine residues of proteins. The enzyme does neither respond to Y, 5' cyclic AMP nor to Y, 5' cyclic GMP. The substrate ATP stabilizes the enzyme against heat inactivation.

The protein kinase activity is subjected to changes during the development of the cell. The specific activity increases during the early stages of development, reaches a maximum just prior to cap formation and declines thereafter with further maturation. Lowest specific activity was found in mature A. major cells.

U n s u p p l e m e n t e d Seawater as Culture M e d i u m for Acetabularia

I~ANS-GEORG SCHWEIGER a n d HoI~sT KRETSCHMER

Max-Planck-Institut ftir Zellbiologie, D-2940 Wilhelmshaven, Federal Republic of Germany

Since the early days of cell biological investigation on Acetabularia the so-called Erd- Schreiber medium (FSyn) has been used as the preferred culture medium. This medium is seawater supplemented by phosphate, nitrate and an earth extract. Although a number of attempts were undertaken to improve this solution or even to replace it by an artificial medium none of them proved satisfactory. Preliminary experiments indicated that unsupple- mented seawater might replace the complex and poorly defined Erd-Schreiber medium if provided in a flow-through system. This was demonstrated in the following apparatus: A total of 8 glass tubes with an inner diameter of 20 mm and a length of 500mm were vertically mounted and connected in series. Each tube was divided into 5 equal chambers by 2 cm layers of glass wool. Each chamber was filled with a population of 14 cells, i.e., 70 ceils per tube or a total of 560 cells. The length of the cells at the beginning of the experiment was 14mm. Seawater was pumped through the series of tubes at a speed of 10 ml/hour. After 40 days of such growth conditions, 10 percent of the control cells grown in petri dishes with Erd-Schreiber medium had formed caps while the percentage in tube 1 and 2 was 100 and in tube 3 and 4 was 80. However, in the seawater system cells had a shorter than normal stalk length. These results indicate that seawater initially contains all the ingredients which Acetabularia needs to grow at the rate observed in Erd-Schreiber medium.

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Per inuc lear D e n s e Bodies in Ace tabular ia : S o m e Ul t ras t ruc tura l and C y t o c h e m i c a l Aspec t s

H . SPRING, W. W. FRANKE, H . FALK, a n d S. BERGER

Deutsches Krebsforschungszentrum, Heidelberg, Lehrstuhl fiir Zellbiologie, Institut fiir Biologie II, Universit~it Freiburg i. Br., and Max-Planck-Institut fiir Zellbiologie, Wilhelmshaven, Federal Republic of Germany

The primary nucleus of various species of Acetabularia is surrounded by numerous (up to 20,000) perinuclear dense bodies, which vary in size from 0.2 ~tm to 5 ~tm. The total volume of these bodies can approach values of as much as 300/o of the whole nuclear volume. They reveal a granular part (granule diameter from 20-40nm) and a more centrally located fibrillar part. Frequently, specific membraneous structures and poly- ribosomes are recognized in the periphery of these bodies. A series of cytochemical reactions usually considered to be specific for DNA, e.g., acridine orange, Feulgen reaction, B~NHAtD'S (1969) EDTA differential staining and bleaching procedure showed positive reactions suggesting the occurrence of DNA in these bodies. Other cytochemical experiments such as staining with ethidium bromide and the procedure of MoYNr (1973) gave no clear results.

BERNHARD, W., 1969: J. Ultrastruct. Res. 27, 250. MOYNt, G., 1973: J. Ultrastruct. Res. 43, 102.

D e m o n s t r a t i o n of Act ive Nuc leo lar C i s t rons of the Ace tabu lar ia N u c l e u s w i th the S p r e a d i n g T e c h n i q u e

M. TRENDELENBURG, H . SPRING, U . SCHZER, a n d W. W. FRANliZ

Deutsches Krebsforschungszentrum, Heidelberg, Federal Republic of Germany

In vivo transcribed strands of deoxyribonucleoprotein that contain cistrons for ribosomal RNA are identified in positively stained spread preparations from nuclei and nucleoli isolated from primary nuclei of the two species, Acetabularia mediterranea and A. major. These nuclei contain large aggregates of distinct nucleolar subunits in which regions, covered with lateral fibrils of increasing length ("matrix units"), the presumed cistrons for rRNA, alternate with fibril-free "spacer" intercepts. The lengths of the matrix units which are normally distributed with mean peak lengths of 1.84 ~tm in A. mediterranea and 1.77 ~xm in A. major indicate that only very little RNA, if any, is iost during the processing of the precursors of rRNA. The spacer lengths of the intercepts are less sharply limited with mean peak values of 0.96 ~tm (A. mediterranea) and 1.05 ~tm (A. major) and range from 0.1 btm to 1.4 um in A. med. and from 0.6 ~tm to 1.8 ~tm in A. major, respectively. In addition, larger matrix units of two different size classes (2.8-5.0 btm and 5-12 ~tm) are noted as interspersed and associated with the smaller ones as well as in distinct aggregate units. The data indicate an intensive transcription and a dramatic amplification of rDNA in the growing nucleus during the postgermination phase.


Presence of an Aux in -Like Substance in Ace tabu lar ia

T. VANDEN DRIESSCHE and V. DELEGHER-LANGOHR

Laboratoire de Cytologic et Embryologie mol&ulaires et Laboratoire de Physiologic V6g6tale, Universit6 Libre de Bruxelles, Belgique

In order to determine whether or not auxin-like substances are present in Acetabularia, the method described by NITSCH and NITSCH (Plant Physiol. 31, 94, 1956) and modified by D~L~GHER (Ann. Physiol. vdg6t. Univ. Brux. 8, 113, 1963) has been used. Acetabularia cells have been homogenized and extracted as for auxins. After chromatography, 20 fractions were assayed for biological activity (Nitsch Test). A highly significant peak was consistently found next to but moving slightly faster than commercial IAA. Acetabularia thus contains an auxin-like substance which is not identical to IAA. However, the Substance could be related to IAA since (1) Acetabularia is known to contain considerable amounts of indole compounds (TANDLER 1963) and (2) exogenous 14C-IAA has been found to bind to the membrane fraction of Acetabularia, as shown both by biochemical analysis and autoradiography. The binding occurs as well in the apical as in the basal half of the alga. Identification must await chemical determination. But, interestingly, the binding of exogenous IAA to the membranes of Acetabularia depends on the integrity of the translational machinery of the cell.

Circadian R h ythm of Photosynthes i s as Inf luenced by A n t i - A u x i n

TH~RI~SE VANDEN DR1ESSCHE and MICHJ~LE HAYET

Laboratoire de Cytologic et Embryologie mol6culaires, Universit6 Libre de Bruxelles, Belgique

The phase sensitivity of the circadian rhythm of photosynthesis to morphactins (20 ~tg/mI) is demonstrated by a pattern change of the rhythm of the treated algae as compared with the controls. Only whole anucleate algae have been studied. Since the morphactins are known to interfere with the intracellular movement of IAA in higher plants, it has been assumed that they interfere, in Acetabularia, with endogenous auxins at particular sites (VANDEN DRIESSCH~, Protoplasma, in press). The presence of an IAA-like substance has been evidenced in Acetabularia (VAND~N DRI~SSC~E and D~LEGHER-LANGOHR, this Symposium).

Presence of c - A M P in Ace tabu lar ia

T. VANDEN DRIESSCHE, W. ]V[OENS, and R. KRAM

Laboratoire de Cytologic et Embryologie mol6culaires, Universit6 Libre de Bruxelles, Belgique

3', 5', cyclic AMP, the mediator of many hormones in animals has also been shown to be present in higher plants as well as in Euglena and Chlamydbmonas. However, its role in the mechanism of action of plant hormones still remains controversial.

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The presence of an auxin-like substance in Acetabularia prompted us to investigate whether or not the alga also contains c-AMP, c-AMP was purified by chromatography on Dowex 50 column according to MANGANIELLO and VAUCMAN (Proc. natl. Acad. Sci. 69, 269, 1972) and measured with G*LMaN'S assay (ibid. 67, 305, 1970). Control determinations on phosphodiesterase digested extracts demonstrate the presence of significant levels of c-AMP in Acetabularia.

Nucleolar Fine Structure in Acetabularia mediterranea

C. L. F. WOODCOCK

Department of Zoology, University of Massachusetts, Amherst, MA 01002, U.S.A.

Individual nuclei isolated from cells of A. mediterranea were broken mechanically, or lysed with detergent, and the contents prepared for electron microscopy by the method developed by MiLLEt and BEATTy (Science 164, 955, 1969) for visualizing transcription. Using this technique, several different types of structure were seen which could be correlated with synthetic processes within the nucleus. Chromatin strands showing the characteristic "Christmas tree" arrangement of attached fibrils were presumably ribosomal cistrons on which rRNA was being synthesized in the nucleolar core. An extensive synthesis of rRNA on a large number of ribosomal cistrons might be expected in a nucleolus which supplies rRNA to a giant cell. A second distinct type of structure was chains of ribosome-sized particles which were often aggregated into dense clumps. These may represent a stage in the processing of ribosomes since the particles are morphologically similar to those seen in thin sections of the nucleolar cortex. Also, chromatin fibers which are probably not of nucleolar origin were observed. (Supported in part by a grant from the Research Council, University of Massachusetts.)

Regulation of the Heteromorphic Life Cycle of the Siphonale Green Alga Derbesia-Halicystis

K. ZETSCHE and M. WuTz

Botanisches Institut der Universit~it, D-6300 Giessen, Federal Republic of Germany

During the life cycle of the green marine alga Derbesia rnarina-Halicystis ovalis a diploid filamentous sporophyte alternates with a haploid ball-like gametophyte called Halicystis. Besides this wild type we have a so called mutant and a parthenogenetic form at our disposal. The mutant is a haploid form. Its morphology is very similiar to those of the wild type Derbesia and its zoospores grow out directly to a Derbesia-like form. The parthenogenetic form looks also like Derbesia. Biochemically sporophyte and gametophyte are characterized by a different cell wall composition and enzyme pattern. The main polysaccharide of the cell wall of the sporophyte (Derbesia) is a mannan while the cell wall polysaceharides of the gametophyte are composed mainly of glucose and xylose. The cell wall composition of the mutant is


similar to that of D erbesia. On the other hand the composition of the cell wall of the parthenogenetic form is, to a certain degree, similar to both Derbesia and Halicystis. Differences in enzyme pattern exist especially in the enzymes of GDPM--and mannan synthesis. These enzymes are present in the Derbesia wild type, the mutant and in the parthenogenetic form but are absent or present only in weak activities in Halicystis. The question arises whether these differences are due to different degree of ploidy in the two generations or the separation of unequal chromosome pairs during reduction division. Our findings exclude these two possibilities. We suggest that the differences are brought about by a relatively stable pattern of gene activity which is only changed during the formation of zoospores and the formation or fusion of gametes.

third symposium onacetabularia

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