A temperature-conditional protoplast of Chlamydomonas reinhardi

PRELIMINARY

NOTES

Printed in Sweden Copyrighr @ 1978 by Academic Press, Inc. All rights of reproduction in any form reserved 0014-4827/78/1172-0439%02.00/0

A temperature-conditional protoplast of Chlamydomonas reinhardi R. LOPPES and R. DELTOUR, Laboratories of Molecular Genetics and Plant Morphology, Department of Botany, University of Litge, Sart Tilman, B-4000 Likge, Belgium Summary. A mutant of the green alga Chlamydomonas reinhardi has been isolated which forms a cell

wall at 25°C but not at 35°C. This conditional protoplast might be of interest in all studies where the temporary presence or absence of the cell wall is desired. .

Algal protoplasts may be considered as models for approaching problems related to the control of the wall formation in plant cells. Numerous mutants of Chlumydomonas reinhardi defective in the process of cell wall synthesis (CW mutants) have been isolated by Davies and his collaborators [l, 21. These mutants revealed several nuclear and extra-nuclear genes [l, 31 involved in the regulation of the wall formation. The cell wall components of Chlumydomonus wildtype strain may be dissociated and reassembled in vitro. The normal structure appears to be composed of five crystalline layers sandwiched between two amorphous layers. Many cell wall mutants are able to produce the crystalline lattice but presumably lack the inner amorphous layer to which the crystalline layers must be attached [4]. The mode of assembly of the different layers in vivo, however, remains relatively obscure. We thought that the very best tools for investigating this kind of problem would probably be mutant strains in which the cell wall formation could be strictly controlled by environmental conditions. It has been shown recently that Chlumy-

domonas

cell wall mutants could be rapidly detected by a specific staining reaction for phosphatase activity [5]. We made use of this method to isolate a thermosensitive cell wall mutant.

Materials and Methods The strain P2Pa derived from the 137 c wild-type strain and lacking the two constitutive acid phosphatases [6] was used in this study. Like the WT strain, P,P, produces both neutral and alkaline derepressible phosphatases when grown on a medium deprived of inorganic phosphate (-P medium) [7]. (+P) and (-P) medium are derived from the Tris minimal phosphate medium given by Surzycki [8]. The first one contains 5 mM inorganic phosphate, the second one is deprived of phosphate. For solid media 15 g Difco agar were added per liter. Cells were grown in continuous light (5 000 lux) at 25°C or 35°C. Colonies were stained for phosphatase activity using a mixture of 1 mg/ml Na-naphthylphosphate (Merck) and 2 mg/ml tetrazotized-o-dianisidine (Sigma) in 0.05 M acetate buffer, pH 4.8. The mutant was isolated after treatment of P,P, cells for 30 min with 50 pg/ml N-methyl-N’nitro-N-nitrosoguanidine (MNNG) in 0.02 M potassium phosphate buffer, pH 7.0,22”C. Survival was about 10% in these conditions. The genetic analysis was carried out according to the method of Levine & Ebersold [9]. For electron microscopy. the cells were fixed with 4% glutaraldehyde in O.-l-M sodium cacodylate buffer pH 7.0, for 2 h, then post-fixed with 2% osmic acid in water for 5 h. The samples were dehydrated through an alcohol series and embedded in Epon. Sections were post-stained according to Reynolds [lo].

Results and Discussion

The derepressible phosphatases produced by the wild-type strain are located partly inside (in the cell wall and between the wall and the plasmalemma [7]), partly outside the cells. They are fully released in the surrounding medium by the cell wall mutants [ 111. The thermosensitive cell wall mutants should have the following phenotype ,with respect to phosphatase activity: staining of the colonies and of,the:medium when cells are grown at low temperature (25°C) staining of the culture medium only when cells are grown at high temperature (35°C). Exp Cd Res 117 11978)

440

Preliminury notes

Fig. 1. Sections through cells of CW,, grown for 5 days on minimal medium at 2s”C (a) or 35°C (b). (a) x9600; (b) x7 200.

P,P, cells were treated with MNNG- and plated on (+P) medium. After a lo-day incubation in the light (5000 lux, 25”C), surviving colonies were replica-plated twice on (-P) medium to allow the synthesis of the derepressible phosphatases. One series of plates was placed at 25°C the other one at 35°C. After 4 days, the colonies were stained for phosphatase activity. Those colonies which showed a phosphatase activity when grown at 25”C, but did not when grown at 35°C were isolated and tested for the presence of the enzyme in the culture medium. Out of about 6000 colonies, one mutant (CW,,,) was found which synthesized the phosphatases at 25 and 35°C but released the major part of them at 35°C. The mutant cells were able to divide at 35°C and to form colonies on solid media. CWtsl colonies growing on solid media at 25°C were compact with clear-cut edges while those developing at 35°C had a slightly disorganized aspect and the irregular E.rp Cd Rrs 117 (1978)

edges characteristic of a cell wall mutant [l, 121. Cells of CW,,, grown at 25 or 35°C were prepared for the observation in electron microscopy. The results clearly demonstrate (fig. 1) the absence of cell wall in CW,, grown at 35°C (about 50 cells were examined in each series). Moreover, when CW,,, cells were grown for 5 days at high temperature then transferred for one day at 25°C a wall was visible in most cells which means that the protoplast rapidly turns back to a wild-type morphology at the permissive temperature. It must be stressed that the cell wall was always present in P,P, grown at 35°C. The character modified in CWtsl is coded by a single nuclear gene as indicated by the fact that zygotes formed by crossing the mutant with WT segregated wild and mutant phenotypes in a 1: 1 ratio. The CW,,, mutant is, to our knowledge, the first thermosensitive cell wall mutant

Preliminary

isolated in Chlamydomonas. It can be considered as a conditional protoplast able to rapidly lose or regenerate its cell wall by simple transfers from one temperature to another. Contrary to non-conditional cell wall mutants, mutants like CW,,, could be specifically used to study in vivo the sequence of events involved in the cell wall formation. After prolonged growth at 35°C returning the protoplasts to the permissive temperature would allow the different steps of the wall formation to be investigated by various methods.

notes

441

conventional micromanipulator. The micropipette is located in a hole through the optical axis of the condenser lens of a normal microscope. The three-dimensional movement of the micropipette is simply controlled by manipulation of the adjusting screws controlling the movement of the condenser. In comparison with conventional microinjection instruments, the simplicity of this new instrument is outstanding.

Since 1859, when Schmidt first tried to manipulate an amoeba with his hand-made manipulator [ 11, micromanipulating or microinjecting techniques have been improved [2]. Recently, Graessmann [3] and Diacumakos [4] developed microinjection apparatuses, by which intracellular injecR. L. is Chercheur qualitiic du Fonds National Belge tion or nuclear transplantation can be perde la Recherche Scientifique. formed in mammalian culture cells. All microinjecting techniques using a micropiReferences pette have so far had to depend on the aid 1. Davies, D R & Plaskitt, A, Genet res 17 (1971) 33. 2. Hyams, J & Davies, D R, Mutation res 14 (1972) of micromanipulators. This communication 351. introduces a new simple method of micro3. Davies, D R, Mol gen genet 115 (1972) 334. 4. Davies, D R & Roberts, K, Genetics of algae (ed R injection without using a micromanipulator. A Lewin) p. 63. Blackwell Scientific Publications, According to this method, the only equipOxford (1976). ment needed for microinjection with a mi5. Loppes, R & Deltour, R, Plant sci lett 8 (1977) 261. 6. Loppes, R & Matagne, R F, Genetics 75 (1973) cropipette is a normal light microscope with 593. 7. Matagne, RF, Loppes, R & Deltour, R, J bact 126 a modified condenser. One principal ad(1976) 937. vantage of the method in comparison with 8. Surzycki, S, Methods in enzymology (ed A San Pietro) vol. 23, part A, p. 67. Academic Press, the conventional microinjecting technique New York (1971). is the simplicity of the apparatus which it9. Levine, R P & Ebersold, W T, Cold Spring Harbor self is easy to construct and operate. symp quant bio123 (1958) 101. 10. Reynolds, E S, J cell biol 17 (1%3) 208. 11. Loppes, R, J bact 128 (1976) 114. 12. Gresshoff, PM, Austj plant physio13 (1976) 457.

Equipment for microinjection

Instead of a micromanipulator a rebuilt condenser is used to hold the micropipette. A perpendicular hole, about 1.5 mm in diaPrinted in Sweden meter, can be made through the optical axis Copyright @ 1978 by Academic Press, Inc. of the condenser lens. A glass micropipette All rights of reproduction in any Form reserved 0014-4827/78/1172-0441~2.00/0 is inserted into the hole (fig. 1). The microA simple microilljection technique not pipette is fixed with a stop, the tip of which employing a micromanipulator can be set about 5 mm from the upper surFUMIICHIRO YAMAMOTO and MITSURU FU- face of the condenser lens. The lower end RUSAWA, Laboratory of Embryology, Department of the micropipette projecting from the botof Biology, Faculty of Science, Osaka City University, tom of the condenser is joined with a plastic Osaka 558, Japan tubing connected to a syringe (see fig. 1). Summary. A new microinjection method is presented in which a modified condenser is used instead of a When the condenser is placed in the micro-

Received June 14, 1978 Revised version received September 7, 1978 Accepted September 7, 1978