Clinostation influence on regeneration of cell wall in Solanum Tuberosum L. protoplasts

Adv. SpaceRes. Vol. 14, No. 8, pp. (8)97-(8)101.1994 Copyright © 1994 COSPAR

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CLINOSTATION INFLUENCE ON REGENERATION OF CELL WALL IN SOLANUM TUBEROSUM L. PROTOPLASTS Elena M. Ncdukha,*,** V. A. Sidorov*** and V. M. Samoylov*** ** Institute of Botany, Academy of Sciences of Ukraine, Tereschenkovskaya sir. 2, 252004 Kiev, Ukraine *** Institute of Cell Biology and Genetic Engineering, Academy of Sciences of Ukraine, Tereschenkovskaya str. 2, 252004 Kiev, Ukraine

ABSTRACT Regeneration of cell walls in protoplasts was investigated using light- and electronmicroscopic methods. The protoplasts were isolated from mesophyll of Solauum tuberosum leaves and were cultivated on the horizontal low rotating ol'inostat (2 rpm) and in control for 10 days. Using a fluorescent method (with Calcofluor white) it was demonstrated that changes in vector gravity results in an regeneration inhibition of cell wall. With electronmicroscopical and electro-cytochemical methods (staining with alcianum blue) dymamics of the regeneration of cell walls in protoplasts was studied! carbohydrate matrix of cell walls is deposited at the earliest stages of this process. The influence of microgravity on the cell wall regeneration is discussed in higher plants. INTRODUCTION It has been previously shown /1/ that growth disturbances as well as changes in the structure of cytoplasmic organelle~ and cell walls occur in cells of higher plants grown in space. Enzymes of the pectolytic complex and calcium i~ns are involved in the mechanism of these changes /2,3/. We have assumed that changes in ultrastructure of cell walls are mediated also by breaches in the polysaccharides synthesis. To confirm this assumpti~ on, we have studied the regeneration of cell wall in Solanum tuberosum L. protoplasts cultivated in an horizontal clinostat, which can reproduce the biological effects of microgravity /3,1/. Regeneration of the cell wall is known to depend on the method of isolation of protoplasts, culturing and species-specificity /4,5/. METHODOLOGY Protoplasts were isolated from the mesophyll of leaves of Solanum tuberosum L__~.plants grown aseptically in test tubes. Leaves were cut up in small pieces and placed for 12-14 hours^in a medium consisting in a mixture of enzymes in osmotic solution at +20uC (I g leaves/10 ml solution): 0,5% Onozsuka R, 0,2% Cellulosin, 0,5% Macerosyme R, 0,5% Saccharose, 5 mM CaCI 9. The suspension of protoplasts was filtered through a nylon net with 50mI00 e~e~smesho Protoplasts were washed in the W-5 solution according the method cribed by Sidorov et el. /6/. Prot~plasts were grown in Petri-dishes (diam. 3,5 x I sm) for 10 days at +24vC in an horizontal clinostat (2 rpm) and exposed for 16 hours to fluorescent white light (1OOO lux) and 8 hours dakness for. Regenerating cell walls of the protoplasts were identified by means after staining with 0,01% calcofluor white in 0 , 1 M phosphate buffer pH 7,2 for 5-10 min and washed in solution a buffer according Herth /7/. Protoplaste were examined by luminescent microscopy (ML-2). Luminescence was induced by exposure to blue light from mercurial lamp with light filters SZS + FS; the length wave of the luminescence excitation was 475-485 nm. Percentage of protoplasts (n=300) with regenerated cell walls was calculated each 24 hours during 10 days. Dimensions of protoplasts (n=100) were calculated with ocular and object-micrometers using a microscope MBB-IA. The protoplasts were fixed for light optical microscopy according the same procedure. Naked proto~lasts were prefixed in I% glutaraldehyde in solution W-5 for I h at +20 C. Protoplasts cultivated on clinostat and in control in solid medium S-V-S were fixed in I% glutaraldehyde in 0,05 M caoodylate buffer, pH 7,0 for 2 h at room temperature, washed in medium W-5 or in a cacodylate buffer and examined by phase-contrast microscope MBB-IA. Naked protoplasts were prefixed for electron microscopy in I% glutaraldehyde in the W-5 solution for I h fixed in 2,5 % glutaraldehyde ~To whom all correspondence should be addressed (8)97

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E.M. Nedukha et al.

for 3 h at 20 °, and washed in the W-5 solution for 15 min. Protoplasts were cultivated under static conditions clinostating and control were prefixe~ by 2,5% glutaraldehyde in 0,05 M cacodylate buffer (pH 7,2) for 4 h at 20~C, after washing in an identical buffer for 15 ~in they were postfixed in I% OsO 4 in a cacodylate buffer for 12 h at +4vC, pH 7,2. Dehydration and embedding in epon-araldite resins were carried out according the usual methods. Ultra-thin sections were studied an using electron microscope JEOL 1200 Xo The matrix polysaccharides in cell walls was stained with 0,05-0,1% solution of alcianum blue added to the prefixing solution of glutaraldehyde for electron-cytochemical microscopy. RESULTS AND DISCUSSION Control Light-optical study of Solanum tuberosum protoplasts isolated from mesophyll of potato leaves and grown in control variant has shown that they have a spherical shade (Fig° 1,a), with a mean diameter of 16 um. Large chloroplasts are equally distributed in the whole volume of protoplasts. Measurements confirm that 10 hours after isolation of protoplasts, their size remains unchanged; after 48 hours the size of protoplasts increases but ~m..dise~eet manner (Table I); 72 hours later it increases more than 3 times and, by the 10th day, the mean size is 80 x 35)~m

Fig. I. Protoplasts of Solanum tuberosum L. a - naked protoplasts; b - protoplasts cultivated in the S-V-S medium for 48 hours, 72 hours (c,f), for 96 hours (d). Fluorescent material in the wall cellulose was stained by calcofluor white after 7 days cultivation (e); a-e - control, f - clinostat. Barsffi20}~n TABLE I Size of Solanum tuberosum protoplasts cultured on the horizontal clinostat (2rpm) and in control in the solid S-V-S medium. Results are the means o~ three experiments. Each size represents the means~SE protoplasts in each experiment Hours (h) and days (d) of cultivation

Size of protoplasts, Control

micrometers

Clinostat

0,1 h (naked protoplasts)

16!I

16±1

2 d

21~1

2~±1

3 d 4 d 7 d 10 d

55~4 6011 66~4 80~2

x x x x

35#1 36!1 35±2 3521

36~2 41±2 44~2 x 36~I 4611 x 36~I

The shape of the protoplasts changes during cultivation: from spherical it becomes oval or elongated (Fig. 1,b-d). The appearance of luminescence in the region of cell wall formation has been observed using the calcofluor white which is a specific dye for cellulose. It was established that only 2% protoplasts showed bluish luminescence on the periphery of protoplasts after 48 hours of cultivation, while a very intensive luminescence was

Clinostation on Cell Wall P.~gcncrafion

observed 6 days later. After lOth day the number of luminescent wall reached 42% of the total number of (Tabl. 2). It can be noted that luminescence of the lized (spot-like) site of the protoplast extending (Fig. 1,e).

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protoplasts with a cultivated protoplasts wall appears in a localater to its whole

TABLE 2 Percentages of Solanum tuberosum in which cell wall was regenerated (n=300) pr°t°plasts Days of cultivation

Percentage Control

1 2 4 6 8 10

0 2 5 10 21 42

of

protoplasts Clinostat 0 1 3 4 4 10

Electron-microscopical examination of ultra-thin sections of naked protoplasts has shown that the mixture of enzymes entirel~ destroys the cell wall, the cytoplasma leaving unchanged (Fig. 2, a-c). The plasmalemma is observed clearly. Chloroplasts are either oval or round shaped, their size is 5-6 x 1,5-2)~m; they have a grana structure, thylakoids are not clear.

Fig. 2. Protoplasts of Solanum tuberosum L. Controls. a-c - naked protoplasts; protoplasts cultivated for 24 hours (d) and 48 hours (e). Abbreviations: P1 - plasmalemma, Ch - chloroplast, M - mltochondrion, LB - lipid body, N - nucleus; bars=1 um There are many osmiophylic lipid bodies in the cytoplasm. The endoplasmic reticulum is of smooth type and made of short cisternes. Mitochondria are round shaped, their diameter is about 0,5Jum; cristae have a small size and matrix is electron-transparent. Golgian structures are not observed. The electron-transparent hyaloplasm contains lipid bodies with a diameter ranging from 0,2 to 0,5~Am; some bodies are connected to the tonoplast of vacuoles. A nucleus is laciniate-shaped in section, it forms not much deep blades, the perinuclear space is uniform at all extent (about 40 run). The whole nucleoplasm is electron-transparent; a compact chromatin is not

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E.M. Nedukha et al.

observed, granular and fibrillar components are present in a nucleus. After a 24 hour culturing the hyaloplasm of protoplasts becomes more denser, particulary near the plasmalemma, where ribosomes and rough endoplasmic reticulum are seen (Fig. 2,d). The granal structure in chloroplasts is c l e a ~ The structure of other crganelles remaining unchanged. 48 hours later starch grains and plastoglobules appear in chloroplasts (Pig. 2,e) as well as recently developed rough endoplasmic reticulum. Two types of chromatin are observed in nucleus: a dense type attached to the nucleus envelope and spread form. The nucleolus is large (near S,3jtun), with distinct granular and fibrillar components. 72 h later, single microfibrills of different length (Fig. 3.a,b) and small vesicular structures which after staining with alcianum blue exhibit a mode rate electron density and fine-granular content are observed on the surface ~of protoplasts. The electron-dense content of the wall in several places irregulary covers the prctoplast, Long channels of the rough endoplasmic reticulum are observed near the plasmalemma zone. Their membranes can be connected to the plasmalemma. The Golgi apparatus is represented by clusters of short cisternas and vesicles (20 nm in diam.) . Mitochondria are of mean size (0,5 x 0,Sjum) and have an elepsoid shape; cristae are more or less long, matrix is electron-dense. Plastids contain large starch grains. Seven days later, the cell wall covers the whole surface of protoplast (Pig. 3,c) but its thickness is not constant and ranges from 0,05 to 0,2 }~m. There are fibrillar structures but main part of the wall is made up of a fine-granular electron-dense content (staining with alcianum blue). The granular structure of the wall gradually becomes very clean; in same some protoplasts the wall thickness is 0,5 p~n and the wall exhibits a finegranular content.

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Fig. 3- Fragments oflpotato protoplasts cultivated for 72 hours (a, b) and 7 days in control (c) and $ days too on horizontal clinostat (d). Abbreviations: Mf - cellulose microfibrils, W - cell wall, ER - endoplasmic reticulum, V - vesicular structure, AG - structure of Apparatus Golgi; bars=ljum Clinostation The study of Solanum tuberosum protoplasts grown on horizontal clinostat has shown t h a ~ - - ~ growth for 48 hour cultivation was quite similar to the control. The size of protoplasts increased slightly as well as in the control. However, 72 hours later their sizes increased (Table I), but less than in control. As the 10th day, sizes was the mean 4 6 ~ m for the long axis and 36.um for the short one. The shape of protoplasts changed according

Clinostation on Cell Wall Regeneration

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their growth; spherical in a first stage, elongated or ellipsoidal later (Fig. 1,f). Luminescent microscopy of calcofluor-stained protoplasts has allowed to demonstrate that the regeneration of cell walls occured only in some protoplasts. At the 10th day, the lumSnescent content of protoplasts was 10% (with a regenerated wall) (Table 2). Ultrastructural study of protoplasts cultivated on the clinostat has shown that regeneration of their cell wall follows the same way as the control but the wall regeneration occurs one day later, Ultrastructure of protoplasts at the 96th h looks like that in protoplasts at the 72th h. In 10 day~ the surface of protoplasts is covered with a layer of cell wall which consisted of electron dense matrix in which microfibriles are spread out in many directions. Most protoplasts (90%) cultivated in clinostat did not regenerate the cell wall and died. Thus, light-optical and electron microscopical study of protoplasts isolated from mescphyll Solanum tuberosL~n leaves and cultivated in vitro has shown that the shape, size and ultrastructure change and similar for many points to other species of plants previosly described /4, 5/. Application of calcofluor which forms durable bonds with cellulose molecules /7/ in the wall permitts to suppose that polysaccharide assembly begins in the definite part of plasmalemma. The inhibition of cell wall regeneration in protoplasts cultivated on the clinostat (Table 2) can occur, due to different exogenous and endogenous factors. Recently a direct correlation has been established between the wall regeneration and plasmalemma fluidity /8/. That suggests the possibility of decrease in the plasmalemma fluidity and disturbances of its transport functions in plant cells under conditions of clinostating. We can assume that certain changes occur also ih~ the synthesis and/or in the transport of the monosaccharides required for the synthesis of microfibrils and pclysaccharides of c e l l w a l l matrix. Data of electron microscopy confirm the role of the ~rough endoplasmic reticulum was in cell wall regeneration as it was previously reported in eucaryotic protoplasts /4, 5/; it plays a the major role in formation of cell vesicles but not the Golgi apparatus. We have stressed the electron-dense matrix of the wall during its generation using the alcianum blue staining technic, which makes unsoluble complex With the free carboxyl groups of polysaccharides /9/. It is known that inhibition of cell wall regeneration is also inhibited by cycloheximide which blocks the protein synthesis, affects the plasmalemma actin receptor and destroys the actin-filaments /8/. It is obvious that clinostating is followed by the disappearance of cytoplasmic movement and transport of vesicular structures required for a normal construction of the cell wall. REFERENCES I. K.M.Sytnik, E.L. Kordyum, E.M. Nedukha et al. Plant cell with variation of geophysical factors, Naukova Dumka, Kiev (1984). 2. E.M. Nedukha, I.A. Trutneva. Role of pectinases in the mechanism of protonema cell wall changes of moss under clinostating, Dokl. Acad. Sci. Ukr. SSR, Ser. B, N 7, 71-74(1988). 3. E.M. Nedukha, Effects of clinostating on disturbution of calcium ions in Funaria hygrometrica moss protonema cells, Dokl. Acad. Sci. Ukr. SSR, S e r . - - ~ - ~ 4 ~ 9 ). 4. E.C. Cocking, Plant cell protoplast-isolation and development, Ann. Rev. Plant Physiol. 23, 29-50 (1972). • 5. J.P. Latge, J. Eilenberg, A. Beauvais st al., Morphology of Entomophtora muscae protoplasts grown in vitro, Protoplasms, 146, 166-173 (1988). 6. V.A. Sidorov, N.N. Piven, Ju. Gleba, K.M. Sytnik, Somatic hybridization of Solanaceae, Naukova Dumka, Kiev (1985). 7. W. Herth, E. Schnepf, The fluorochrome calcofluor white, binds oriented to structural polysaccharide fibrils, Protoplasma, 105, 129-133 (1980). 8. R. Legge, R.M. Brown, Modification of protoplast cell wall regeneration by membrane perturbation, Protoplasma, 143, 38-42 (1988). 9. G. ~uintarelli, J.E. Scott, M.C. Dellovo, The chemical and histochemical properties of alcian blue.lll. Chemical blocking and unblocking, Histochemie, 4, 99-112 (1964).