Heterogeneity of chloroplasts in Acetabularia mediterranea. Heterogeneous distribution and morphology of chloroplast DNA

Preliminary

notes

483

fluent monolayer then occupied the surface Beezer) pp. 1112. Academic Press, London-New Francisco (1980). area available for cell growth. Under these 3. York-ToronteSydney-San Lamprecht, I, Ibidem, pp. 43-112. conditions the metabolism becomes re- 4. Woledge, R C, Ibidem, pp. 145-162. Kemp, R B, Ibidem, pp. 113-130. stricted to processes of maintenance which 5. 6. NikoliC, D & NeSkoviC, B, Jugoslav physiol pharexhibit only a low but constant heat evolumacol acta 12 ( 1976) 191. D P, Dorsey, J K & Bolen, D W, Biotion. From the plateau phase a value of 7. Cerretti, chim biophys acta 462 (1977) 748. (40flO) pW/cell has been calculated. This 8. Ljungholm, K, Wadso, I & KjellCn, L, Acta path01 Stand, sect. B 86 (1978) 121. figure is in the same order of magnitude 9. microbial Kemp, R B, Pestic sci 6 (1975) 311. as that obtained by other authors for cells Received September 30, 1980 of comparable size but belonging to con- Accepted November 4, 1980 tinuous (‘established’) lines (table 1, items l-4). The only other value available for a finite line (table 1, item 5) is much lower, Copyright 0 1’981 hy Academic Press. Inc. but this may be due to difficulties in keepAll right5 of reproduction in any firm rewvd oil 14.4X27/X I /~1?04X3-0h%0?.~k~/0 ing the cells attached and alive under the Heterogeneity of chloroplasts in applied culture conditions. Our experiments were performed in a Acetabuluria mediterranea. Heterogeneous non-stirred and non-aerated batch vessel, distribution and morphology of chloroi.e. under non-optimum aerobic conditions. plast DNA Nevertheless, the metabolism does not A. LDTTKE, Department of Radiobiology, Nuclear seem to be oxygen limited, since occasional Research Centre, 2400 Mol. Belgium removal of the vessel’s cap and gentle stir- Summary. A high nercentage of chloroplasts in the ring of the medium did not cause increased siphonaceous g&en alga Acetabularia mediterranea lacks DNA: staining with the sensitive DNA-soecific heat production. fluorochrome 4’-6-diamidino-2-phenylindole CDAPI) In conclusion, the foil-culture technique revealed that DNA was present only in 47-51% of total plastid population. The distribution of DNAused in our experiments results in repro- the containinn chloronlasts aooeared heterogeneous, exducible heat profiles. Its main advantage hibiting & apicobasal gmdient. DNA could be dein 64. 46. 35. and 36% of the olastids from the over other procedures described in the tected apical, subapical, middle, and basal-part of the cell, literature is (i) that rather sensitive cells can respectively. DNA morphology also exhibited heteroThree types of nucleoid were distinguished: be kept alive and free of microbial conta- geneity. (1) round compact nucleoids; (2) long thin nucleoids mination in the calorimetric vessel for sev- characteristic of chloroplasts of the cell apex and the and (3) elaborate nucleoids appearing to coneral days without agitation and aeration; whorls; sist of several subunits, which were-more typical of and (ii) that the progress of cell develop- the middle and basal part of the cell. On the basis of the nucleoid morphoiogy and the decrease in DNAment may be followed, if necessary, micro- containing plastids from the apex towards the basis scopically by removing the seeded foil of the cell, we propose a model for the development plastids lacking DNA in relation to chloroplast from and reinserting it into the calorimeter of replication. vessel during the experiments. Attempts to apply the method in screening thera- Several ultrastructural [l-6] and biochemical [ 1, 7, 83 investigations on chloropIast peutic agents are in progress. DNA of the giant green unicellular and uninucleate alga Acetabularia mediterranea References have shown that this DNA has unique fea1. Spink, C & Wads& I, Methods in biochemical analysis (ed D Glick) vol. 23, pp. l-159. Wiley tures. Unlike chloroplast DNA of Euglena Interscience, New York (1976). Chlamydomonas reinhardi, and 2. Belaich, J P, Biological microcalorimetry (ed A D gracilis, Printed

in Sweden

3lt-801810

Exp Cell Res 131 (1981)

484

Preliminary

notes

several higher plants [9], circular molecules falling into the size of 40-45 pm have not been found in DNA preparations isolated by conventional methods [lo, 1l] or by molecular sieving [4], nor after gentle lysis of the plastids [IO] or disruption by osmotic shock [l-3]. Length measurements made on purified chloroplast DNA as well as on DNA released after osmotic shock cover thebroadrangeof5-20OOpm[ld, 10,111. Although the precise size and conformation of the DNA are still unknown, it is generally accepted that this plastome is exceptionally large. According to renaturation kinetics the genetic complexity is about one order of magnitude larger than that of other algae and thus of the same order of magnitude as in bacterial cells [S]. A lack of chloroplast DNA in 65-80% of the total plastid population had been reported for A. mediterranea and Polyphysa (Acetabularia) cliftonii [3] as well as for A. calyculus, A. crenulata, and the closely related Dasycladacea Batophora oerstedii

rm Since all determinations on the percentage of plastids with DNA were average values, while other plastid features (size, morphology, photosynthetic activity, fluorescence emission, starch content) were shown to vary along an apico-basal gradient (for review, see [ 13]), we reinvestigated the distribution of chloroplast DNA not only in the total chloroplast population but also in the chloroplast, populations of different parts of the cell. Special attention was paid to the arrangement of the nucleoids in the plastids by using the sensitive DNA-specific fluorochrome 4’-6-diamidino-2-phenylindole [ 141. The data on the distribution of chloroplast DNA within the cell together with the morphological appearance of the nucleoid is the basis of the proposed model for the Exp Cell Res 131 (1981)

development of DNA-less plastids in relation to chloroplast division. Materials

and Methods

Cultures of Acetabularia mediterranea were grown according to Lateur [IS] in “Erdschreiber” medium under normal conditions (21°C; 12: 12 h light/dark cycle: annrox. 1000 lux). Exneriments were nerformed with algae at stages 4’ and‘7 according to’ the code given by Bonotto & Kirchmann [ 161. For staining of chloroplast DNA (cpDNA) with the DNA soecific fluorochrome 4’-6diamidino-2-ohenvlindole iDAPI, kindly provided by Professor 0: Da&, University of Erlangen, BRD) chloroplasts were isolated by either one of the outlined methods. Method 1. Acetabularia mediterranea cells were fried according to Franke et al. [17] in 6% glutaraldehyde (0.1 M phosphate buffer, pH 7.4) for 1 h at room temperature, after which the cells were cut into 0.5-l cm pieces and the fixation continued for 6-12 h in fresh glutaraldehyde. Upon transfer of the fragments into SSC, the cytoplasm was expelled out of the cell wall and floated on the buffer surface. The cytoplasmic fragments were incubated with DAPI (40 pg/ml) for 2 h, washed 3X in SSC, and mounted in SSC for viewing. Method II. After brushing and removal of the rhizoid, whole cells or 5 mm fragments were gently homogenized in Erdschreiber medium with 15% glucose and 4% formaldehyde. After filtration of the slurry through four layers of cheesecloth or passage through a 12 pm nucleopore filter, the fotation of the plastids was continued for at least 0.5 h. Chloroplasts were pelleted at 500 g and the pellet was washed 2x with SSC. The plastids were incubated with DAPI (2 pg/ml) for 12 h, washed 2x with SSC, and mounted for viewing in the same buffer. Fluorescent microsconical observations were made with a Zeiss photomi&oscope equipped with Neofluor DhaSe obiectives and a HBO 200-W He lamp. DNAiDAPI fluorescence was examined with & UG:l excitation filter in combination with a 418 nm barrier filter, while the autofluorescence of chlorophyll was monitored with a BG-12 excitation filter in combination with a 500 nm barrier filter. All preparations for the determination of olastid DNA were examined at both falter combinations. Photographs were taken on Kodak Tri-X pan with exposure times of 0.5-2 min; fdm development was nerformed with Microdol X diluted 1 : 3. For the eniargement of single plastids the negatives were rephotographed on Kodak high contrast copy fdm under an ordinary binocular and then contact copied on Kodak Kodalith ortho type 3 2556.

Results

Staining of cytoplasmic fragments (method I) with the DNA specific fluorochrome DAPI clearly demonstrated the characteristic blue fluorescence of the DNA/DAPI

Preliminary

notes

485

Fig. I. Chloroplasts from A. mediterranea isolated and stained with DAPI according to method I. (a) Note the different sizes and brightness of the DNA/DAPI

fluorescence (<). The arrow indicates a type 1 nucleoid. (b) The same preparation, showing the autofluorescence of the chlorophyll. x3 300. Bar, 5 pm.

complex in localized regions in some of the plastids. An example of this type of preparation is given in fig. 1. The fluorescence of chloroplast DNA is confined to a small area (fig. la) in contrast to the overall red

fluorescence of the plastids (fig. lb), as demonstrated by the same group of plastids. The number of plastids exhibiting DNA fluorescence was counted in suspensions prepared by method II from cells at two

Table 1. DAPZjluorescence

Table 2. DAPZfluorescence in chloroplasts of apical, subapical, middle and basal fragments of Acetabularia mediterranea

in chloroplasts of whole cells of Acetabularia mediterranea from three separate experiments

Ceil type

No. No. with counted DNA

% with DNA

Vegetative, 25 mm stalk Vegetative, 2 mm cap Vegetative, 2 mm cap

174 174 91

47 49 51

82 86 46

No. with DNA

%

Fragment type

No. counted

Apical Subapical Middle Basal

210 190 227 249

121 97 80 89

z 35 36

with DNAIE

Exp Cell Res 131 (1981)

486

Preliminary notes

ferences were observed in the intraorganellar DNA (i.e. nucleoid) arrangement. Three I types were distinguished: (1) in small round : plastids, the DNA appears to occupy almost the whole volume of the organelle (arrow, fii. la); (2) in long thin plastids the DNA is seen to extend throughout the organelle in a longitudinal direction (fig. 2); (3) in large plastids with one or more starch grains one or more elaborate DNA centres were present (fig. 3). Types 1 and 2 were mainly found in the apical part and the whorls of the cell. Due to photographic accentuation of the nucleoid the plastid’s morphology is not visible, but indicated on the print by a white line. Type 3 chloroplast morphology and the localization of the nucleoid between one Fig. 2. Nucleoid of a long chloroplast (type 2), which starch grain on the left and a row of three appears to consist of three units connected with each grains on the right is demonstrated in fig. other. The chloroplast morphology, not visible on the print, is indicated by a white line. Isolation and stain- 3 a. Underexposure of the same plastid (fig. ing according to method II. x5700. Bar, 1 pm. 36) revealed several ring-like subunits of the nucleoid substructure. The large type 3 nucleoids with interspersed unstained areas developmental stages. Under our culture were frequently observed in plastids of the conditions DNA was detectable in 47, 49, middle and basal part of the cell. and 51% of the total chloroplast populaDiscussion tion, respectively (table 1). Different proportions of plastids with and The lack of DNA in part of the chloroplast without DNA are found in samples derived population of Acetabularia mediterranea from selected parts of the cell (table 2). agrees with earlier investigations on the In samples isolated from the uppermost same and some other Acetabufaria species tip including the whorls 64% of the chloro- as well as on the related Dasycladacea plasts exhibited DNA fluorescence, while Batophora oerstedii [3, 121. Yet, up to the the percentage was 46, 35 and 36% in sam- present no attempt has been made to exples isolated from the subapical, middle, plain the unusual occurrence of plastids and basal part of the cell, respectively. without DNA. In this discussion we exClose inspection of several chloroplast elude the physiological implications, but preparations showed noticeable differences merely focus on the question of the mode in llPe fluorescence intensity of the DNA/ of derivation of ‘DNA-less’ plastids from DAPI complex as well as in the number plastids containing DNA in cells grown of nucleoids per plastid (70% with one under normal culture conditions. nucleoid, 28% with two nucleoids and 2% For the increase in ‘DNA-less* plastids in with four nucleoids). Also, remarkable dif- the apico-basal direction concomitant with Exp Cell Res I31 (1981)

Preliminary

notes

487

Fig. 3. Chloroplast with a nucleoid of type 3. (a) One can discern the plastid’s morphology and the localization of the nucleoid spaced between one starch grain on the left and a row of three grains to the

right; (b) The substructure of the nucleoid consisting of ring-like subunits becomes discernible upon underexposure of the same chloroplast. X5700. Bar, 5 pm.

a change in the nucleoid morphology, we propose that structural features of the cpDNA and thylakoid arrangements are re-

sponsible for the development of ‘DNAless’ plastids during the course of chloroplast growth and replication. On the basis of our microscopical observations on the nucleoid morphology, as well as on the lack of DNA in part of the plastid population, and on the mode of plastid replication described [19] we propose a model for the distribution of DNA along an apico-basal gradient as depicted in fig. 4. In small round plastids filled with DNA (fig. 4a) or in long plastids with an extended DNA region, mainly found in the apical part of the cell, the nucleoid replicates and is pushed apart by the growing diagonal lamellae prior to chloroplast division (fig. 4b, c). In the middle and basal part of the cell, where chloroplast division is less frequent [20], asymmetric growth of the plastids results in a large organelle consisting of two or more basic morphological entities and one nucleoid acentrically located (fig. 4d). Equal division of such an organelle could consequently lead to one plastid with DNA and one plastid without DNA (fig. 4e). This model explains the formation of plastids without DNA as well as the occurrence of plastids with elaborate DNA

h

d

E.g. 4. Model for the derivation of plastids without DNA from plastids containing DNA. (a) Small plastid with centrally located DNA; (b) replication of the nucleoid concomitant with the growth of the diagonal lamellae; (c) separation of the nucleoid by the inversely growing- diagonal lamellae; (d) asymmetric growth of the chloroplast resulting in an organelle consisting of two morphological entities and one nucleoid acentrically located; (e) equal division of the plastid leading to one organelle with DNA and one organelle without DNA. e, envelope: el, external lamellae; dl, diagonal lamellae; n, nucleoid. 32-801810

Exp CellRes 131 (19&?/J

488

Preliminary

notes

centres, since ongoing DNA synthesis accompanied by asymmetric growth of the plastid results in a large organelle with aggregated DNA. I thank Drs S. Bonotto and M. Janowski for critical reading of the text. It would have been impossible to produce figs 2 and 3 without the patient help of Dr W. Baeyens. I thank Mrs Cecile Huysmans for preparing the manuscript. Research was supported by the European Communities and the CENISCK.

References 1. Green, B R, Burton, H, Heilpom, V & Limbosch, S, Biology of Acetabularia (ed J Brachet & S Bonotto) p. 35. Academic Press, New York (1970). 2. Green, B R & Burton, H, Science 168(1970) 981. 3. Woodcock, C L F & Bogorad, L, J cell biol 44 (1970) 361. 4. Bonotto, S, Lurquin, P F, Baeyens, W, Charles, P, Hoursiangou-Neubrun, D, Mazza, A, Tramontano, G & Felluga, B, Protoplasma 83 (1975) 172. 5. Mazza, A, Bonotto, S & Felluga, B, Progress in Acetabularia research (ed C L F Woodcock) p. 123. Academic Press, New York (1977). 6. Mazza, A, Bonotto, S, Felluga, B, Casale, A & Sassone Corsi. P. Develoomental bioloev of Acetabularia (ed S Bonotio, V Kefeli -h S Puiseux-Dao) p. 115. Elsevier/North-Holland Biomedical Press, Amsterdam (1979). 7. Heiluom. V & Limbosch. S. Eur i biochem 22 (197i) 573. 8. Green, B R, Muir, B L & Padmanabhan, U, Progress in Acetabuluria research (ed C L F Woodcock) p. 107. Academic Press, New York (1977). 9. Bedbrook, J R & Kolodner, R, Ann rev plant nhvsiol30 ( 1979)593. 10. Green, B R; Protoplasma 75 (1972) 478. 11. Werz. G & Kellner. G. J ultrastruct res 24 (1968) . , 109. 12. Coleman, A W, J cell bio182 (1979) 299. 13. Bonotto, S, Lurquin, P F & Mazza, A, Adv marine biol 14 (1976) 123. 14. Schnedl, W, Dann, 0 & Schweizer, D, Eur j cell biol20 (1980) 290. 15. Lateur, L, Rev algol 1 (1973) 26. 16. Bonotto, S & Kirchmann, R, Bull sot roy bot belg 103 (1970) 255. 17. Franke, W W, Berger, S, Falk, H, Spring, H, Scheer, U, Herth, W, Trendelenburg, M F & Schweiger, H G, Protoplasma 82 (1974) 249. 18. Shephard, D C, Exp cell res 37 (1965) 93. 19. Puiseux-Dao, S, Acetabufaria and cell biology. Logos Press (1970). 20. Clauss, H, Liittke, A, Hellman, F & Reinert, J, Protoplasma 69 (1970) 313. .

Received October 31, 1980 Accepted October 31, 1980 Exp Cell Res I31 (1981)

Labelling of the chromatoid body by [“Hluridine in rat pachytene spermatocytes KARL-OVE

SGDERSTRGM, Department

ogy, University

of Patholof Turku, SF-20520 Turku 52, Finland

In this study it is shown that a cytoplasmic cell organelle, the chromatoid body, becomes labelled with 13Hluridine in the pachytene spermatocytes. The chromatoid body becomes labelied when the cells are first labelled for 2 h in the presence of r3H]uridine and thereafter chased for 9 h in the presence of unlabelled uridine. This labelling is inhibited by the specific RNA polymerase II inhibitor o-amanitin. Based on this it is suggested that part of the RNA synthesized in the pachytene spermatocytes is stored in the chromatoid body and transported to the postmeiotic spermatids where it is used in the differentiation of the spermatids.

Summary.

Both in male and female germ cells there exist cytoplasmic ultrastructurally similar cell organelles which in male mammals have been called the chromatoid body [5, 63. It is first seen during spermatogenesis as a distinct cell organelle in mid-pachytene stage and is present in the meiotic divisions and in the post-meiotic cells, the spermatids [ 1, 5, 6, 181. The chemical composition as well as the function of this cell organelle are unknown. Previous light microscopic histochemical data suggest that it contains RNA [2, 211, but electron microscopic histochemistry has failed to confirm this [4]. However, high resolution autoradiography has shown that the chromatoid body becomes labelled with [3H]uridine in the spermatids of the rat [20]. Because the chromatoid body is a clearly recognizable cell organelle already in the mid-pachytene spermatocytes its labelling with r3H]uridine has been studied in these cells. Materials

and Methods

The rats were killed by a blow on the head. The tubules of the testes were prepared free and pieces containing a complete seminiferous epithelial cycle were isolated using the transillumination method [19]. Printed

in Sweden