Planta
Planta (1982)155:97 104
9 Springer-Verlag 1982
Multinucleate cyst formation in Acetabularia mediterranea after x-irradiation A. Lfittke* and S. Bonotto Department of Radiobiology, S.C.K.-C.E.N., Boeretang 200, B-2400 Mol, Belgium
Abstract. Cells of A c e t a b u l a r i a m e d i t e r r a n e a were irradiated with increasing doses of X-rays ( 6 4 . 5 258.10 . 4 kC kg-1). The cells are radioresistant up to 193.5.10 -4 kC k g - t in terms of growth and progression through the life cycle but the morphogenesis of whorls, caps, and cysts is accompanied by morphological alterations. Microscopical examination of cyst bearing caps in irradiated cells has shown the presence of giant cysts neighboring particularly small ones. Photographic recording of cyst development showed that the multinucleate cap cytoplasm partitions into multinucleate portions rather than uninucleate ones as in the control cells. After complete cleavage a cyst wall is deposited onto the multinucleate cytoplasm. In contrast to uninucleate cysts with one lid the wall contains multiple lids. Their number appears to correspond to the number of nuclei in the cytoplasm compartment during cleavage. The results indicate that X-rays preferentially inhibit the synthesis of a factor which plays a role in establishing the normal spatial morphogenetic pattern necessary for cyst formation.
Key words: A c e t a b u l a r i a - Chlorophyta - Cyst formation Nuclei, secondary ( A c e t a b u l a r i a ) - Nucleo-cytoplasmic relations - X-Irradiation.
Introduction
The different stages of the life cycle of the giant green alga A c e t a b u l a r i a m e d i t e r r a n e a 1 (for reviews on the life cycle see Bonotto and Kirchmann 1970; Schweiger and Berger 1979; K o o p 1979) are characInstitut ffir Entwicklungsphysiologie, D-5000 K61n 41 (Lindenthal), Gyrhofstrasse 17, FRG According to the International Code of Botanical Nomenclature the correct name is now Acetabularia acetabulum (L.) Silva. As the name mediterranea has been used in all the published work referred to in this article, it has been retained to avoid confusion
* Present address.:
terized by distinct morphological and biochemical markers (Berger et al. 1974). This feature has been successfully exploited for studying radiation effects on cell differentiation (for review see Bonotto et al. 1976). A survey of the literature shows that both gamma and X-rays cause severe morphological alterations during all developmental stages (Bacq et al. 1955; Bonotto and Bonnijns-Van Gelder 1969; Bonotto et al. 1970; Bonotto and Kirchmann 1972; Bonotto 1975; Bonotto et al. 1979; Bonotto et al. 1980). A m o n g the different processes of differentiation, cyst formation - considering time, a narrow stage within the life cycle - appeared most feasible for further investigations of X-ray induced malformations. During the transition from the vegetative to the generative phase the originally diploid uninucleate cell passes through a multinucleate phase and then produces haploid uninucleate gametangia, the so-called cysts (Schulze 1939; K o o p 1979). In studying the morphogenesis of cysts, Werz (1968a, b) concluded that the rearrangement of the cap-ray cytoplasm is under the control of the secondary nuclei. Some ultrastructural aspects of this process were described by Woodcock (1971) and by Berger et al. (1975). This paper describes the development of giant cysts in cells of A c e t a b u l a r i a which had been irradiated with X-rays during the vegetative phase prior to cap formation.
Materials and methods
Cultures of Aeetabularia mediterranea were grown according to Lateur (1963) in Erd-Schreiber medium (ESM; 21~ C; 12 h photoperiod; 1,000 lx). Cells 17-24 mm long and having no caps yet (see Fig. la), were irradiated with doses from 64.5-258.10 -4 kC kg 1 using a RT 250 Philips X-ray source. Irradiation was performed from below at a distance of 1 cm to the bottom of the plastic Petri dish and using a sample size of 25-40 cells in 30 ml ESM. After irradiation the cells were immediately transferred from the Petri
0032-0935/82/0155/0097/$01.60
A. Lfittke and S. Bonotto: Cyst formation in Acetabularia after X-irradiation
98
CY
Fig. 1 a-d. Scheme for the developmental
I!~ S 21DAYS, ) a ~;H b IRR+ADIATION
AO+BSERVATION
dishes into culture flasks with fresh ESM in order to avoid possible secondary effects by the irradiated medium. Cyst formation was monitored under a binocular microscope. Cells with secondary nuclei in the cap rays ("white s p o t s " stage; see Fig. 1 c; Schulze 1939) were photographed at hourly intervals until cyst formation was completed. For size measurements mature cysts were isolated from the caps and r a n d o m samples photographed under a microscope. The longer axis was measured on prints. The cyst wall was isolated from maturate cysts prior to gamete formation. Cysts were fixed in 6% glutaraldehyde (0.1 M phosphate, pH 7.4) for 24 h. After several washes in phosphate buffer giant cysts were cut into half under a binocular and the cytoplasm squeezed out with the aid of pointed glass needles. The empty cyst wall was examined under a Reichert microscope equipped with Nomarski optics.
100 90 80-
/
,"
I I/ ,,,"
70-
/
60-
i/, /.i
50
.,";
//
,/
/ ,,'
.o /// / /" 30~I/i/' x4rr.'$4'/'/'" '
20
/"
_i-" I
._._..........I ----"-
-'I
..~"
/*
./"
"/
=
10-
/
Results
Figure 1 illustrates the morphogenetic steps of the life cycle of A c e t a b u l a r i a m e d i t e r r a n e a - which is approx. six months long (Bonotto and Kirchmann 1970), which were the subject matter of this investigation. It also shows the time which elapsed between irradiation (Fig. 1 a) and cyst formation (Fig. 1 c, d). Exposure of the cells to single X-ray doses up to 129-10-4kC kg -1 does not prevent cap formation (Fig. 1 b) nor the division of the primary nucleus and the migration of the secondary nuclei into the cap (Fig. 1 c), nor cyst formation (Fig. 1 d). After irradiation with doses up to 129-10 .4 kC kg -1, cap formation, division of the primary nucleus and the subsequent cyst formation are delayed, in comparison to the nonirradiated controls, although the rate of cap development is later restored to almost normal (Fig. 2). The percent of cells developing cysts is also close to that in the controls. However, microscopic examination reveals between the cysts in control and irradiated cells striking differences which are not clearly evident to the naked eye. While the morphology of the cysts in control cells is uniform, that of cysts in irradiated cells is variable. Isolating the cysts from the cap rays (Fig. 3 c, d) and measuring the longer axis allowed a precise comparison. Cysts of control cells fall into the range of 95-155 gm with a Table 1. Kinetics for cyst formation in Acetabularia mediterranea irradiated at a cell length of 17 mm. 30 cells per sample
= A
o-----o B 9 ....... 9 C o---= D
/ ,/
Dose kC kg 1
DAYS
O0
5
1{)
15
2'0
25
3'0
stages of Acetabularia mediterranea investigated and the approximate time from irradiation to cyst development, a Vegetative cell of about 15-25 ram; b cell with nearly mature cap; e cell in the "white s p o t s " stage; d cell after cyst formation. RH, rhizoid; S, stalk; W, whorls; PN, primary nucleus; CA, cap; SN, secondary nuclei; CY, cysts
3'5
Fig. 2. Kinetics for cap formation of A. mediterranea cells irradiated at a cell length of 24 mm. For each dose duplicate samples of 25 cells/30 ml ESM were used. A e - - e 0 kC kg -1 ; B 9 . . . . . 9 129.10 4 k C k g 1 ; C e . . . . . . e 1 9 3 . 5 . 1 0 4 k C k g - 1 ; D *--e 258.10 4 k C k g -1
0 64.5.I0 -* 129 .10 .4 258 "10 -4
N u m b e r of caps with cysts (%) Day 62
D a y 69
D a y 79
Day 107
60 20 27 3
80 40 40 3
97 73 70 7
97 87 87 40
a 1 kC kg-r0.39 .10 ~ kr
A. Ltittke and S. Bonotto: Cyst formation in Acetabularia after X-irradiation
99
Fig. 3a-d. Cysts in situ and after isolation, a Portion of a cap of a control cell; b portion of a cap of an irradiated cell after 44 days of exposure to a single dose of 129.10 -~ kC kg-1; bar equals 200 p.m; e isolated cysts from control cells; d isolated cysts from irradiated cells; bar equals 50 gin. CW, cap ray wall; CY, cyst; CYW, cyst wall
mean of 143 gm, while cysts of cells irradiated with 1 2 9 - 1 0 - 4 k C kg -1 range from 8 3 - 2 0 2 g m with a mean of 131 gm (Fig. 4). It should be noted that in the latter frequency distribution cysts of more than 400 ~tm had not been considered. In order to pinpoint the process of malformation a number of irradiated cells at the white spots stage (Schulze 1939) were continuously monitored during cyst development (see Fig. l c, d). Selected photographs of a series with normal cyst formation taken over 24 h documents the cytoplasmic rearrangement (Fig. 5). The early indication for cyst formation is the occurrence of white spots (arrows) in the cap cytoplasm, which represent the location of secondary
nuclei in chloroplast-free areas (Fig. 5a; 9 h). Two hours later the secondary nuclei are arranged in nearly equidistant position, and each of them is surrounded by a portion of cytoplasm (Fig. 5b; 11.00 h). Within the following four hours the cytoplasm condenses (Fig. 5c; 15.00 h) and briefly afterwards compartition of the cytoplasm around each single nucleus (arrow) is clearly visible (Fig. 5d; 17.20 h). The further partitioning of the cytoplasm rapidly proceeds from the outer cap ray cytoplasm toward the inner one (Fig. 5e, f; 19.00 and 20.00h). During the transition of the cytoplasm from the multinucleate (white spots stage) to the uninucleate stage (cysts) mitotic divisions of the secondary nuclei are arrested. Nuclear divisions
100
A. Ltittke and S. Bonotto : Cyst formation in Acetabularia after X-irradiation
a N=333
Z 0 I--
6
rn n"
2
6
r
18
22
b
Z IaJ 0 LU re LI.
SIZE CLASS
rel.units
Fig. 4a, b. Frequency distribution of the cyst size in control cells (a) and cells after 45 days of exposure to a single dose of 129. t 0-4 kC kg- 1 (b). Cysts of more than 400 gm in the sample from irradiated cells were not considered. N, number of cysts measured; 1 rel. unit equals 11.9gm
start again after the first layers of cyst wall are deposited. For the development of giant cysts two possibilities may exist: for unknown reasons the cytoplasm divided into large irregularly shaped portions around each single nucleus, or otherwise the multinucleate cytoplasm partitioned into multinucleate portions rather than uninucleate ones. The first hypothesis implied a tremendous change in the ratio nucleus: cytoplasm which, according to the uniform cyst size in control cells, appears rather stringently maintained. In addition, the number of secondary nuclei had to be much lower. In the second hypothesis it is anticipated that the number of secondary nuclei is more or less comparable to that in the control ceils, but there is a free-run of the cytoplasm contraction into unequal portions. Close inspection of cyst formation in irradiated cells left no doubt that the latter hypothesis holds as shown with two prints of the same cap out of a series taken over 19 h (Fig. 6). In Fig. 6a white spots (ray 1, 2, 5) are visible as well as already compartmentalized cytoplasm (ray 3, 4). Since the appear-
ance of white spots is due to the presence of secondary nuclei in the cap cytoplasm lying flattened against the wall (Schulze 1939; Godward etal. 1979), this constitutes good proof that meiosis of the primary nucleus in the rhizoid, subsequent mitotic division and the migration of the secondary nuclei into the cap cytoplasm took place (for review on meiosis and mitosis in Acetabularia see K o o p 1979). Figure 6b shows the cap cytoplasm partitioned into large portions with several white spots (and therefore several secondary nuclei) and particularly small ones after 8 h. Cyst formation is nearly completed after 18.5 h and white spots in the giant cysts are barely visible. The positioning of the secondary nuclei was always more difficult to detect in irradiated cells in comparison to the controls. But, there was no indication for further nuclear divisions during the cytoplasm compartmentalization. Also, during this sequence it became obvious that the partitioning of the cytoplasm into the final cyst form did not proceed in apico-basal direction within the rays as usually observed (see Fig. 5e). The microscopical analysis for the development of giant Cyst with several nuclei rather than one was corroborated by examining the cyst wall several days after cyst formation. Cysts having a single secondary nucleus contain only one lid in the thick cyst wall (Fig. 7a), which opens during germination for gamete release (Fig. 7b). An intimate correlation between the position of the original secondary nucleus and the lid had recently been demonstrated by fluorescence microscopy (Zimmer and Werz 1980). In contrast, cyst walls of giant cysts contain numerous lids (Fig. 7c). In general, the larger the cyst the more lids were observed. This strongly suggests the stringent maintainance of the correlation between the position of the secondary nuclei and the lids. Therefore, the number of nuclei included in the cytoplasm compartment during cyst formation ought to be equal to the number of lids in the cyst wall. Discussion Several earlier papers stated strong radioresistance of the giant unicellular alga Acetabularia mediterranea toward UV rays (Hfimmerling 1956; Six 1958) gamma rays (Bonotto et al. 1970; Bonotto and Kirchmann 1972; Bonotto 1975; Bonotto etal. 1979), as well as X-rays (Bacq et al. 1955; Bacq et al. 1957) as far as growth, morphogenesis, and regeneration of rhizoidal fragments is concerned. The morphological alterations of the cells after exposure ot X-rays, described by Bacq et al. (1957) and recently by us for the cell stages outlined in Fig. 1 (Bonotto et al. 1980), suggest, however, a kind of
A. Liittke and S. Bonotto: Cyst formation in Acetabularia after X-irradiation
101
Fig. 5a-f. Photographic recording of cyst development over 11 h. The sequential steps are described for the two rays denoted by arrows. a Early "white spots" stage; b "white spots" stage; secondary nuclei in nearly equidistant position; e condensation of the cytoplasm; tl partitioning of the cytoplasm around each nucleus; e cyst formation starting in the outer part of the cap ray; f cysts formed throughout the ray. CW, cap ray wall; WS, "white spots"; bar equals 500 gm
damage which is not immediately obvious after irradiation, but expressed when growth and differentiation begin. Since cells irradiated with doses up to 129.10-4 kC k g - 1 enter the white spots stage, meiosis of the primary nucleus in the rhizoid and subsequent mitotic divisions (for a recent review on meiosis and mitosis in Aeetabularia see K o o p 1979), as well as the migration of the secondary nuclei into the cap cytoplasm, take place. The presence of cysts, which despite the morphological alterations contain a wall with lids, narrows the discussion to the cyst-forming process itself. W o o d c o c k (1971) has shown the presence of two microtubular systems during cyst morphogenesis. One set of microtubules surrounds the secondary nuclei in the areas which are seen as white spots by light microscopy. The second one radiates from the nuclei into the organelle-containing cytoplasm. Cyst forma-
tion can be arrested by colchicine, when the drug is applied at the early white spots stage (see Fig. 5a), but not a t a later stage (i.e., Fig. 5d; Werz 1968a; Werz 1969 Woodcock 1971). After transfer into drugfree medium, malformed cysts develop resembling those shown here for irradiated cells. Actinomycin D and puromycin given at the early white spots stage cause the same effect, namely inhibition of cyst formation in the presence, differentiation of malformed cysts after removal of the drugs (Werz 1968). Since all three inhibitors were applied after the positioning of the secondary nuclei in the cap ray cytoplasm, the giant malformed cysts formed after lifting the inhibition most likely have arisen from several nuclei in the same way as in irradiated cells. The action of colchicine on preventing microtubular assembly is well known (for review see Dustin 1978), and actinomycin D and puromycin are widely used inhibitors for transcription and translation, respectively. Corn-
102
A. Lfittke and S. Bonotto : Cyst formation in Acetabularia after X-irradiation
Fig. 6a-c. Giant cyst development in a cell irradiated with 129.10 - 4 kC kg -1. a "White spots" stage in rays 1, 2, 5, partitioning of the cytoplasm in rays 3, 4. b Partitioning of large cytoplasm portions with several "white spots" in rays 1, 2, 5 after 8 h. e Giant cysts after 18.5 h. WS, "white spots"; GC, giant cysts; bar equals 500 I~m
A. Ltittke and S. Bonotto: Cyst formation in Acetabularia after X-irradiation
103
Fig. 7a-c. Cyst walls, a Cyst wall of a control cell; the lid is visible upon condensation of the cytoplasm, b Empty cyst wall with open lid after gamete release, c Isolated cyst wall from an irradiated cell with several lids. CW, cyst wall; L, lid; bar equals 25 gm in a and b, and 100 lam in c
b i n i n g the results o f the three i n h i b i t o r s with those r e p o r t e d here it m u s t be c o n c l u d e d t h a t X - r a y s exert an effect o n the m i c r o t u b u l a r systems p r e s e n t d u r i n g cyst f o r m a t i o n ( W o o d c o c k 1971). A t present, we cann o t decide w h e t h e r the synthesis for m i c r o t u b u l a r p r o t e i n or m i c r o t u b u l a r o r g a n i z i n g centers ( M T O C ) is b l o c k e d or if, w h a t seems m o r e likely, the synthesis o f s o m e o t h e r f a c t o r is i n v o l v e d in the f o r m a t i o n o f the c o r r e c t m i c r o t u b u l e p a t t e r n d u r i n g cyst f o r m a tion. F o r the l o n g interval b e t w e e n i r r a d i a t i o n a n d cyst f o r m a t i o n (see Fig. 1), we feel it u n l i k e l y t h a t X - r a y s cause a m e r e d i s o r g a n i z a t i o n o f p r e f o r m e d s t r u c t u r a l elements. I n this case a recycling o f existing structures over several weeks h a d to be a s s u m e d . W e suggest t h a t X - r a y s p r e f e r e n t i a l l y i n h i b i t the synthesis o f a f a c t o r w h i c h p l a y s a role in e s t a b l i s h i n g a n o r m a l s p a t i a l p a t t e r n necessary for cyst m o r p h o genesis. This i n t e r p r e t a t i o n m i g h t e q u a l l y e x p l a i n o t h e r m o r p h o l o g i c a l a l t e r a t i o n s o b s e r v e d after X - i r r a -
d i a t i o n as m a l f o r m e d whorls a n d caps ( B o n o t t o et al. 1980). The f o r m a t i o n o f a r e g u l a r s t r u c t u r a l p a t t e r n initiating the m o r p h o g e n e s i s o f whorls a n d caps seems to be d i s t u r b e d . We acknowledge Mr. A. Bossus for technical assistance and Mrs. M.C. Hemelaers-Gielen for typewriting the text.
References Bacq, Z.M., Damblon, J., Herve, A. (t955) Radiorgsistance d'une algue Acetabularia mediterranea Lamour. Compt. Rend. S6ances Soc. Biol. (Paris) 149, 1512-1515 Bacq, Z.M., Vanderhaeghe, J., Damblon, J., Errera, M., Herve, A. (1957) Effets des rayons X sur AcetabuIaria mediterranea. Exp. Cell Res. 12, 63%648 Berger, S., Sandakhchiev, L., Schweiger, H.G. (1974) Fine structural and biochemical markers of Dasycladaceae. J. Microsc. 19, 8%104 Berger, S., Herth, W., Franke, W.W., Falk, H, Spring, H., Schweiger, H.G. (1975) Morphology of the nucleo-cytoplasmic interactions during the development of Acetabularia cells. Protoplasma 84, 223 256
104
A. Ltittke and S. Bonotto: Cyst formation in Acetabularia after X-irradiation
Bonotto, S. (1975) Morphogenesis in normal, branched and irradiated Acetabularia mediterranea. Pubbl. Staz. Zool. Napoli 39, 9(~ 107 Bonotto, S., Bonnijns-Van Gelder, E. (1969) A simple method to obtain high frequency of stalk division in Acetabularia mediterranea. Plant Physiol. 44, 1738-1741 Bonotto, S., Kirchmann, R. (1970) Sur les processus morphog6nbtiques d'Acetabularia mediterranea. Bull. Soc. R. Bot. Belgique, 103, 255 272 Bonotto, S., Kirchmann, R. (1972) Sur un type particulier de morphogen~se anormale provoqu+e par les radiations gamma chez l'Acetabularia mediterranea. G. Bot. Ital. 106, 21-27 Bonotto, S., Kirchmann, R., Janowski, M., Netrawali, M.S. (1970) Effects of gamma-radiation on Acetabularia mediterranea. In: Biology of Acetabularia, pp. 255-271, Brachet, J., Bonotto S., eds. Academic Press, New York London Bonotto, S., Lurquin, P., Mazza, A. (1976) Recent advances in research on the marine alga Acetabularia. Adv. Mar. Biol. 14, 123 250 Bonotto, S., D'Emilio, M.A., Kirchmann, R. (1979) Recent work on the biological and biochemical effects of gamma radiations on the unicellular marine alga Acetabularia rnediterranea. In: Developmental biology of Acetabularia, pp. 49-64, Bonotto, S., Kefeli, V., Puiseuy-Dao, S., eds. Elsevier/North Holland Biomedical Press, Amsterdam New York Oxford Bonotto, S., Lfittke, A., Hoursiangou-Neubrun, D., Puiseux-Dao, S., Bossus, A., Kirchmann, R. (1980) Effects on X-rays on Acetabularia mediterranea. In : Radiation effects on aquatic organisms, pp. 26%276, Egami, N., ed. Japan Scientific Societies Press, Tokyo; University Park Press, Baltimore Dustin, P. (1978) Microtubules. Springer, Berlin Heidelberg New York Godward, M.B.E., Beth, K., Paccy, J. (1979) Nuclear division in the cyst, and white spot nuclei preceding cyst formation in Acetabularia wettsteinii. Protoplasma 101, 37-46
Hgmmerling, J. (1956) Wirkungen yon UV- und R6ntgenstrahlen auf kernlose und kernhaltige Teile yon Acetabularia. Z. Naturforsch, l l b , 217-221 Koop, H.-U. (1979) The life cycle of Acetabularia (Dasycladales, Chlorophyceae): a compilation of evidence for meiosis in the primary nucleus. Protoplasma 100, 353-366 Lateur, L. (1963) Une technique de culture pour l'Acetabularia mediterranea. Rev. Algol. 1, 26-37 Schweiger, H.-G., Berger, S. (1979) Nucleocytoplasmic interrelationships in Acetabularia and some other Dasycladaceae. Int. Rev. Cytol. Suppl. 9, 11-44 Schulze, K.L. (1939)Cytologische Untersuchungen an Acetabularia mediterranea und Acetabularia wettsteinii. Arch. Protistenkd. 92, 17%225 Six, E. (1958) Die Wirkung yon Strahlen auf Acetabularia. III. Mitt.: Die Wirkung yon RSntgenstrahlen und ultravioletten Strahlen auf kernhaltige Teile yon Acetabularia mediterranea. Z. Naturforsch. 13b, 6-14 Werz, G. (1968a) Plasmatische Formbildung als Voraussetzung fiir die Zellwandbildung bei der Morphogenese von Acetabularia. Protoplasma 65, 81-96 Werz, G. (1968b) Differenzierung und Zeltwandbildung in isoliertern Cytoplasma aus Acetabularia. Protoplasma 65, 349-357 Werz, G. (1969) Morphogenetic process in Acetabularia. In: Inhibitors, tools in cell research,pp. 167-186, B/icher, Th., Sies, H. eds. Springer, Berlin Heidelberg New York Woodcock, C.L.F. (1971) The anchoring of nuclei by cytoplasmic microtubules in Acetabularia. J. Cell Sci. 8, 611-621 Zimmer, B., Werz, G. (1980) Concanavalin A affects polysaccharidic wall formation and mitotic activity in Polyphysa (Acetabularia) cliftonii protoplasts. Exp. Cell Res. 126, 29%310
Received 24 November 1980; accepted 14 April 1981
Planta (1982)155:97 104
9 Springer-Verlag 1982
Multinucleate cyst formation in Acetabularia mediterranea after x-irradiation A. Lfittke* and S. Bonotto Department of Radiobiology, S.C.K.-C.E.N., Boeretang 200, B-2400 Mol, Belgium
Abstract. Cells of A c e t a b u l a r i a m e d i t e r r a n e a were irradiated with increasing doses of X-rays ( 6 4 . 5 258.10 . 4 kC kg-1). The cells are radioresistant up to 193.5.10 -4 kC k g - t in terms of growth and progression through the life cycle but the morphogenesis of whorls, caps, and cysts is accompanied by morphological alterations. Microscopical examination of cyst bearing caps in irradiated cells has shown the presence of giant cysts neighboring particularly small ones. Photographic recording of cyst development showed that the multinucleate cap cytoplasm partitions into multinucleate portions rather than uninucleate ones as in the control cells. After complete cleavage a cyst wall is deposited onto the multinucleate cytoplasm. In contrast to uninucleate cysts with one lid the wall contains multiple lids. Their number appears to correspond to the number of nuclei in the cytoplasm compartment during cleavage. The results indicate that X-rays preferentially inhibit the synthesis of a factor which plays a role in establishing the normal spatial morphogenetic pattern necessary for cyst formation.
Key words: A c e t a b u l a r i a - Chlorophyta - Cyst formation Nuclei, secondary ( A c e t a b u l a r i a ) - Nucleo-cytoplasmic relations - X-Irradiation.
Introduction
The different stages of the life cycle of the giant green alga A c e t a b u l a r i a m e d i t e r r a n e a 1 (for reviews on the life cycle see Bonotto and Kirchmann 1970; Schweiger and Berger 1979; K o o p 1979) are characInstitut ffir Entwicklungsphysiologie, D-5000 K61n 41 (Lindenthal), Gyrhofstrasse 17, FRG According to the International Code of Botanical Nomenclature the correct name is now Acetabularia acetabulum (L.) Silva. As the name mediterranea has been used in all the published work referred to in this article, it has been retained to avoid confusion
* Present address.:
terized by distinct morphological and biochemical markers (Berger et al. 1974). This feature has been successfully exploited for studying radiation effects on cell differentiation (for review see Bonotto et al. 1976). A survey of the literature shows that both gamma and X-rays cause severe morphological alterations during all developmental stages (Bacq et al. 1955; Bonotto and Bonnijns-Van Gelder 1969; Bonotto et al. 1970; Bonotto and Kirchmann 1972; Bonotto 1975; Bonotto et al. 1979; Bonotto et al. 1980). A m o n g the different processes of differentiation, cyst formation - considering time, a narrow stage within the life cycle - appeared most feasible for further investigations of X-ray induced malformations. During the transition from the vegetative to the generative phase the originally diploid uninucleate cell passes through a multinucleate phase and then produces haploid uninucleate gametangia, the so-called cysts (Schulze 1939; K o o p 1979). In studying the morphogenesis of cysts, Werz (1968a, b) concluded that the rearrangement of the cap-ray cytoplasm is under the control of the secondary nuclei. Some ultrastructural aspects of this process were described by Woodcock (1971) and by Berger et al. (1975). This paper describes the development of giant cysts in cells of A c e t a b u l a r i a which had been irradiated with X-rays during the vegetative phase prior to cap formation.
Materials and methods
Cultures of Aeetabularia mediterranea were grown according to Lateur (1963) in Erd-Schreiber medium (ESM; 21~ C; 12 h photoperiod; 1,000 lx). Cells 17-24 mm long and having no caps yet (see Fig. la), were irradiated with doses from 64.5-258.10 -4 kC kg 1 using a RT 250 Philips X-ray source. Irradiation was performed from below at a distance of 1 cm to the bottom of the plastic Petri dish and using a sample size of 25-40 cells in 30 ml ESM. After irradiation the cells were immediately transferred from the Petri
0032-0935/82/0155/0097/$01.60
A. Lfittke and S. Bonotto: Cyst formation in Acetabularia after X-irradiation
98
CY
Fig. 1 a-d. Scheme for the developmental
I!~ S 21DAYS, ) a ~;H b IRR+ADIATION
AO+BSERVATION
dishes into culture flasks with fresh ESM in order to avoid possible secondary effects by the irradiated medium. Cyst formation was monitored under a binocular microscope. Cells with secondary nuclei in the cap rays ("white s p o t s " stage; see Fig. 1 c; Schulze 1939) were photographed at hourly intervals until cyst formation was completed. For size measurements mature cysts were isolated from the caps and r a n d o m samples photographed under a microscope. The longer axis was measured on prints. The cyst wall was isolated from maturate cysts prior to gamete formation. Cysts were fixed in 6% glutaraldehyde (0.1 M phosphate, pH 7.4) for 24 h. After several washes in phosphate buffer giant cysts were cut into half under a binocular and the cytoplasm squeezed out with the aid of pointed glass needles. The empty cyst wall was examined under a Reichert microscope equipped with Nomarski optics.
100 90 80-
/
,"
I I/ ,,,"
70-
/
60-
i/, /.i
50
.,";
//
,/
/ ,,'
.o /// / /" 30~I/i/' x4rr.'$4'/'/'" '
20
/"
_i-" I
._._..........I ----"-
-'I
..~"
/*
./"
"/
=
10-
/
Results
Figure 1 illustrates the morphogenetic steps of the life cycle of A c e t a b u l a r i a m e d i t e r r a n e a - which is approx. six months long (Bonotto and Kirchmann 1970), which were the subject matter of this investigation. It also shows the time which elapsed between irradiation (Fig. 1 a) and cyst formation (Fig. 1 c, d). Exposure of the cells to single X-ray doses up to 129-10-4kC kg -1 does not prevent cap formation (Fig. 1 b) nor the division of the primary nucleus and the migration of the secondary nuclei into the cap (Fig. 1 c), nor cyst formation (Fig. 1 d). After irradiation with doses up to 129-10 .4 kC kg -1, cap formation, division of the primary nucleus and the subsequent cyst formation are delayed, in comparison to the nonirradiated controls, although the rate of cap development is later restored to almost normal (Fig. 2). The percent of cells developing cysts is also close to that in the controls. However, microscopic examination reveals between the cysts in control and irradiated cells striking differences which are not clearly evident to the naked eye. While the morphology of the cysts in control cells is uniform, that of cysts in irradiated cells is variable. Isolating the cysts from the cap rays (Fig. 3 c, d) and measuring the longer axis allowed a precise comparison. Cysts of control cells fall into the range of 95-155 gm with a Table 1. Kinetics for cyst formation in Acetabularia mediterranea irradiated at a cell length of 17 mm. 30 cells per sample
= A
o-----o B 9 ....... 9 C o---= D
/ ,/
Dose kC kg 1
DAYS
O0
5
1{)
15
2'0
25
3'0
stages of Acetabularia mediterranea investigated and the approximate time from irradiation to cyst development, a Vegetative cell of about 15-25 ram; b cell with nearly mature cap; e cell in the "white s p o t s " stage; d cell after cyst formation. RH, rhizoid; S, stalk; W, whorls; PN, primary nucleus; CA, cap; SN, secondary nuclei; CY, cysts
3'5
Fig. 2. Kinetics for cap formation of A. mediterranea cells irradiated at a cell length of 24 mm. For each dose duplicate samples of 25 cells/30 ml ESM were used. A e - - e 0 kC kg -1 ; B 9 . . . . . 9 129.10 4 k C k g 1 ; C e . . . . . . e 1 9 3 . 5 . 1 0 4 k C k g - 1 ; D *--e 258.10 4 k C k g -1
0 64.5.I0 -* 129 .10 .4 258 "10 -4
N u m b e r of caps with cysts (%) Day 62
D a y 69
D a y 79
Day 107
60 20 27 3
80 40 40 3
97 73 70 7
97 87 87 40
a 1 kC kg-r0.39 .10 ~ kr
A. Ltittke and S. Bonotto: Cyst formation in Acetabularia after X-irradiation
99
Fig. 3a-d. Cysts in situ and after isolation, a Portion of a cap of a control cell; b portion of a cap of an irradiated cell after 44 days of exposure to a single dose of 129.10 -~ kC kg-1; bar equals 200 p.m; e isolated cysts from control cells; d isolated cysts from irradiated cells; bar equals 50 gin. CW, cap ray wall; CY, cyst; CYW, cyst wall
mean of 143 gm, while cysts of cells irradiated with 1 2 9 - 1 0 - 4 k C kg -1 range from 8 3 - 2 0 2 g m with a mean of 131 gm (Fig. 4). It should be noted that in the latter frequency distribution cysts of more than 400 ~tm had not been considered. In order to pinpoint the process of malformation a number of irradiated cells at the white spots stage (Schulze 1939) were continuously monitored during cyst development (see Fig. l c, d). Selected photographs of a series with normal cyst formation taken over 24 h documents the cytoplasmic rearrangement (Fig. 5). The early indication for cyst formation is the occurrence of white spots (arrows) in the cap cytoplasm, which represent the location of secondary
nuclei in chloroplast-free areas (Fig. 5a; 9 h). Two hours later the secondary nuclei are arranged in nearly equidistant position, and each of them is surrounded by a portion of cytoplasm (Fig. 5b; 11.00 h). Within the following four hours the cytoplasm condenses (Fig. 5c; 15.00 h) and briefly afterwards compartition of the cytoplasm around each single nucleus (arrow) is clearly visible (Fig. 5d; 17.20 h). The further partitioning of the cytoplasm rapidly proceeds from the outer cap ray cytoplasm toward the inner one (Fig. 5e, f; 19.00 and 20.00h). During the transition of the cytoplasm from the multinucleate (white spots stage) to the uninucleate stage (cysts) mitotic divisions of the secondary nuclei are arrested. Nuclear divisions
100
A. Ltittke and S. Bonotto : Cyst formation in Acetabularia after X-irradiation
a N=333
Z 0 I--
6
rn n"
2
6
r
18
22
b
Z IaJ 0 LU re LI.
SIZE CLASS
rel.units
Fig. 4a, b. Frequency distribution of the cyst size in control cells (a) and cells after 45 days of exposure to a single dose of 129. t 0-4 kC kg- 1 (b). Cysts of more than 400 gm in the sample from irradiated cells were not considered. N, number of cysts measured; 1 rel. unit equals 11.9gm
start again after the first layers of cyst wall are deposited. For the development of giant cysts two possibilities may exist: for unknown reasons the cytoplasm divided into large irregularly shaped portions around each single nucleus, or otherwise the multinucleate cytoplasm partitioned into multinucleate portions rather than uninucleate ones. The first hypothesis implied a tremendous change in the ratio nucleus: cytoplasm which, according to the uniform cyst size in control cells, appears rather stringently maintained. In addition, the number of secondary nuclei had to be much lower. In the second hypothesis it is anticipated that the number of secondary nuclei is more or less comparable to that in the control ceils, but there is a free-run of the cytoplasm contraction into unequal portions. Close inspection of cyst formation in irradiated cells left no doubt that the latter hypothesis holds as shown with two prints of the same cap out of a series taken over 19 h (Fig. 6). In Fig. 6a white spots (ray 1, 2, 5) are visible as well as already compartmentalized cytoplasm (ray 3, 4). Since the appear-
ance of white spots is due to the presence of secondary nuclei in the cap cytoplasm lying flattened against the wall (Schulze 1939; Godward etal. 1979), this constitutes good proof that meiosis of the primary nucleus in the rhizoid, subsequent mitotic division and the migration of the secondary nuclei into the cap cytoplasm took place (for review on meiosis and mitosis in Acetabularia see K o o p 1979). Figure 6b shows the cap cytoplasm partitioned into large portions with several white spots (and therefore several secondary nuclei) and particularly small ones after 8 h. Cyst formation is nearly completed after 18.5 h and white spots in the giant cysts are barely visible. The positioning of the secondary nuclei was always more difficult to detect in irradiated cells in comparison to the controls. But, there was no indication for further nuclear divisions during the cytoplasm compartmentalization. Also, during this sequence it became obvious that the partitioning of the cytoplasm into the final cyst form did not proceed in apico-basal direction within the rays as usually observed (see Fig. 5e). The microscopical analysis for the development of giant Cyst with several nuclei rather than one was corroborated by examining the cyst wall several days after cyst formation. Cysts having a single secondary nucleus contain only one lid in the thick cyst wall (Fig. 7a), which opens during germination for gamete release (Fig. 7b). An intimate correlation between the position of the original secondary nucleus and the lid had recently been demonstrated by fluorescence microscopy (Zimmer and Werz 1980). In contrast, cyst walls of giant cysts contain numerous lids (Fig. 7c). In general, the larger the cyst the more lids were observed. This strongly suggests the stringent maintainance of the correlation between the position of the secondary nuclei and the lids. Therefore, the number of nuclei included in the cytoplasm compartment during cyst formation ought to be equal to the number of lids in the cyst wall. Discussion Several earlier papers stated strong radioresistance of the giant unicellular alga Acetabularia mediterranea toward UV rays (Hfimmerling 1956; Six 1958) gamma rays (Bonotto et al. 1970; Bonotto and Kirchmann 1972; Bonotto 1975; Bonotto etal. 1979), as well as X-rays (Bacq et al. 1955; Bacq et al. 1957) as far as growth, morphogenesis, and regeneration of rhizoidal fragments is concerned. The morphological alterations of the cells after exposure ot X-rays, described by Bacq et al. (1957) and recently by us for the cell stages outlined in Fig. 1 (Bonotto et al. 1980), suggest, however, a kind of
A. Liittke and S. Bonotto: Cyst formation in Acetabularia after X-irradiation
101
Fig. 5a-f. Photographic recording of cyst development over 11 h. The sequential steps are described for the two rays denoted by arrows. a Early "white spots" stage; b "white spots" stage; secondary nuclei in nearly equidistant position; e condensation of the cytoplasm; tl partitioning of the cytoplasm around each nucleus; e cyst formation starting in the outer part of the cap ray; f cysts formed throughout the ray. CW, cap ray wall; WS, "white spots"; bar equals 500 gm
damage which is not immediately obvious after irradiation, but expressed when growth and differentiation begin. Since cells irradiated with doses up to 129.10-4 kC k g - 1 enter the white spots stage, meiosis of the primary nucleus in the rhizoid and subsequent mitotic divisions (for a recent review on meiosis and mitosis in Aeetabularia see K o o p 1979), as well as the migration of the secondary nuclei into the cap cytoplasm, take place. The presence of cysts, which despite the morphological alterations contain a wall with lids, narrows the discussion to the cyst-forming process itself. W o o d c o c k (1971) has shown the presence of two microtubular systems during cyst morphogenesis. One set of microtubules surrounds the secondary nuclei in the areas which are seen as white spots by light microscopy. The second one radiates from the nuclei into the organelle-containing cytoplasm. Cyst forma-
tion can be arrested by colchicine, when the drug is applied at the early white spots stage (see Fig. 5a), but not a t a later stage (i.e., Fig. 5d; Werz 1968a; Werz 1969 Woodcock 1971). After transfer into drugfree medium, malformed cysts develop resembling those shown here for irradiated cells. Actinomycin D and puromycin given at the early white spots stage cause the same effect, namely inhibition of cyst formation in the presence, differentiation of malformed cysts after removal of the drugs (Werz 1968). Since all three inhibitors were applied after the positioning of the secondary nuclei in the cap ray cytoplasm, the giant malformed cysts formed after lifting the inhibition most likely have arisen from several nuclei in the same way as in irradiated cells. The action of colchicine on preventing microtubular assembly is well known (for review see Dustin 1978), and actinomycin D and puromycin are widely used inhibitors for transcription and translation, respectively. Corn-
102
A. Lfittke and S. Bonotto : Cyst formation in Acetabularia after X-irradiation
Fig. 6a-c. Giant cyst development in a cell irradiated with 129.10 - 4 kC kg -1. a "White spots" stage in rays 1, 2, 5, partitioning of the cytoplasm in rays 3, 4. b Partitioning of large cytoplasm portions with several "white spots" in rays 1, 2, 5 after 8 h. e Giant cysts after 18.5 h. WS, "white spots"; GC, giant cysts; bar equals 500 I~m
A. Ltittke and S. Bonotto: Cyst formation in Acetabularia after X-irradiation
103
Fig. 7a-c. Cyst walls, a Cyst wall of a control cell; the lid is visible upon condensation of the cytoplasm, b Empty cyst wall with open lid after gamete release, c Isolated cyst wall from an irradiated cell with several lids. CW, cyst wall; L, lid; bar equals 25 gm in a and b, and 100 lam in c
b i n i n g the results o f the three i n h i b i t o r s with those r e p o r t e d here it m u s t be c o n c l u d e d t h a t X - r a y s exert an effect o n the m i c r o t u b u l a r systems p r e s e n t d u r i n g cyst f o r m a t i o n ( W o o d c o c k 1971). A t present, we cann o t decide w h e t h e r the synthesis for m i c r o t u b u l a r p r o t e i n or m i c r o t u b u l a r o r g a n i z i n g centers ( M T O C ) is b l o c k e d or if, w h a t seems m o r e likely, the synthesis o f s o m e o t h e r f a c t o r is i n v o l v e d in the f o r m a t i o n o f the c o r r e c t m i c r o t u b u l e p a t t e r n d u r i n g cyst f o r m a tion. F o r the l o n g interval b e t w e e n i r r a d i a t i o n a n d cyst f o r m a t i o n (see Fig. 1), we feel it u n l i k e l y t h a t X - r a y s cause a m e r e d i s o r g a n i z a t i o n o f p r e f o r m e d s t r u c t u r a l elements. I n this case a recycling o f existing structures over several weeks h a d to be a s s u m e d . W e suggest t h a t X - r a y s p r e f e r e n t i a l l y i n h i b i t the synthesis o f a f a c t o r w h i c h p l a y s a role in e s t a b l i s h i n g a n o r m a l s p a t i a l p a t t e r n necessary for cyst m o r p h o genesis. This i n t e r p r e t a t i o n m i g h t e q u a l l y e x p l a i n o t h e r m o r p h o l o g i c a l a l t e r a t i o n s o b s e r v e d after X - i r r a -
d i a t i o n as m a l f o r m e d whorls a n d caps ( B o n o t t o et al. 1980). The f o r m a t i o n o f a r e g u l a r s t r u c t u r a l p a t t e r n initiating the m o r p h o g e n e s i s o f whorls a n d caps seems to be d i s t u r b e d . We acknowledge Mr. A. Bossus for technical assistance and Mrs. M.C. Hemelaers-Gielen for typewriting the text.
References Bacq, Z.M., Damblon, J., Herve, A. (t955) Radiorgsistance d'une algue Acetabularia mediterranea Lamour. Compt. Rend. S6ances Soc. Biol. (Paris) 149, 1512-1515 Bacq, Z.M., Vanderhaeghe, J., Damblon, J., Errera, M., Herve, A. (1957) Effets des rayons X sur AcetabuIaria mediterranea. Exp. Cell Res. 12, 63%648 Berger, S., Sandakhchiev, L., Schweiger, H.G. (1974) Fine structural and biochemical markers of Dasycladaceae. J. Microsc. 19, 8%104 Berger, S., Herth, W., Franke, W.W., Falk, H, Spring, H., Schweiger, H.G. (1975) Morphology of the nucleo-cytoplasmic interactions during the development of Acetabularia cells. Protoplasma 84, 223 256
104
A. Ltittke and S. Bonotto: Cyst formation in Acetabularia after X-irradiation
Bonotto, S. (1975) Morphogenesis in normal, branched and irradiated Acetabularia mediterranea. Pubbl. Staz. Zool. Napoli 39, 9(~ 107 Bonotto, S., Bonnijns-Van Gelder, E. (1969) A simple method to obtain high frequency of stalk division in Acetabularia mediterranea. Plant Physiol. 44, 1738-1741 Bonotto, S., Kirchmann, R. (1970) Sur les processus morphog6nbtiques d'Acetabularia mediterranea. Bull. Soc. R. Bot. Belgique, 103, 255 272 Bonotto, S., Kirchmann, R. (1972) Sur un type particulier de morphogen~se anormale provoqu+e par les radiations gamma chez l'Acetabularia mediterranea. G. Bot. Ital. 106, 21-27 Bonotto, S., Kirchmann, R., Janowski, M., Netrawali, M.S. (1970) Effects of gamma-radiation on Acetabularia mediterranea. In: Biology of Acetabularia, pp. 255-271, Brachet, J., Bonotto S., eds. Academic Press, New York London Bonotto, S., Lurquin, P., Mazza, A. (1976) Recent advances in research on the marine alga Acetabularia. Adv. Mar. Biol. 14, 123 250 Bonotto, S., D'Emilio, M.A., Kirchmann, R. (1979) Recent work on the biological and biochemical effects of gamma radiations on the unicellular marine alga Acetabularia rnediterranea. In: Developmental biology of Acetabularia, pp. 49-64, Bonotto, S., Kefeli, V., Puiseuy-Dao, S., eds. Elsevier/North Holland Biomedical Press, Amsterdam New York Oxford Bonotto, S., Lfittke, A., Hoursiangou-Neubrun, D., Puiseux-Dao, S., Bossus, A., Kirchmann, R. (1980) Effects on X-rays on Acetabularia mediterranea. In : Radiation effects on aquatic organisms, pp. 26%276, Egami, N., ed. Japan Scientific Societies Press, Tokyo; University Park Press, Baltimore Dustin, P. (1978) Microtubules. Springer, Berlin Heidelberg New York Godward, M.B.E., Beth, K., Paccy, J. (1979) Nuclear division in the cyst, and white spot nuclei preceding cyst formation in Acetabularia wettsteinii. Protoplasma 101, 37-46
Hgmmerling, J. (1956) Wirkungen yon UV- und R6ntgenstrahlen auf kernlose und kernhaltige Teile yon Acetabularia. Z. Naturforsch, l l b , 217-221 Koop, H.-U. (1979) The life cycle of Acetabularia (Dasycladales, Chlorophyceae): a compilation of evidence for meiosis in the primary nucleus. Protoplasma 100, 353-366 Lateur, L. (1963) Une technique de culture pour l'Acetabularia mediterranea. Rev. Algol. 1, 26-37 Schweiger, H.-G., Berger, S. (1979) Nucleocytoplasmic interrelationships in Acetabularia and some other Dasycladaceae. Int. Rev. Cytol. Suppl. 9, 11-44 Schulze, K.L. (1939)Cytologische Untersuchungen an Acetabularia mediterranea und Acetabularia wettsteinii. Arch. Protistenkd. 92, 17%225 Six, E. (1958) Die Wirkung yon Strahlen auf Acetabularia. III. Mitt.: Die Wirkung yon RSntgenstrahlen und ultravioletten Strahlen auf kernhaltige Teile yon Acetabularia mediterranea. Z. Naturforsch. 13b, 6-14 Werz, G. (1968a) Plasmatische Formbildung als Voraussetzung fiir die Zellwandbildung bei der Morphogenese von Acetabularia. Protoplasma 65, 81-96 Werz, G. (1968b) Differenzierung und Zeltwandbildung in isoliertern Cytoplasma aus Acetabularia. Protoplasma 65, 349-357 Werz, G. (1969) Morphogenetic process in Acetabularia. In: Inhibitors, tools in cell research,pp. 167-186, B/icher, Th., Sies, H. eds. Springer, Berlin Heidelberg New York Woodcock, C.L.F. (1971) The anchoring of nuclei by cytoplasmic microtubules in Acetabularia. J. Cell Sci. 8, 611-621 Zimmer, B., Werz, G. (1980) Concanavalin A affects polysaccharidic wall formation and mitotic activity in Polyphysa (Acetabularia) cliftonii protoplasts. Exp. Cell Res. 126, 29%310
Received 24 November 1980; accepted 14 April 1981
