Plant Cell Reports (1983) 2:257-260

Plant Cell Reports © Springer-Verlag 1983

Influence of Growth Conditions on the Composition of Cell Wall Polysaccharides from Cultured Tobacco Cells W. B l a s c h e k a n d G. F r a n z Department of Chemistry and Pharmacy, University of Regensburg, D-8400 Regensburg, FRG Received July 12, 1983 - Communicated by M. H. Zenk

Abstract The sugar composition of c e l l wall p o l y s a c c h a r i d e s of two tobacco v a r i e t i e s obtained from mesophyll, regene r a t i n g p r o t o p l a s t s and c e l l s grown under v a r i o u s cond i t i o n s were compared. Regenerating p r o t o p ] a s t s developed an unusual cell wal] with a low cellulose and a high non-cellulosic glucan content, in the presence of different phytohormones compact and friable ca1]i were obtained with cell walls containine low and high arabinose/xylose ratios. The cell wal]s of compact cal]i were comparable to those of genuine mesophyll cells. The sugar constituents of cell walls obtained from ceils grown in liquid media were different from those of solid calli. The cell wall composition of suspension cultured cel]s was hardly affected by various combinations of phytohormones, but was a]tered by high osmolarity of the medium. Abbreviations: NAA = 1-naphtha]eneacetic acid; 6-BA = 6-benzyl-aminopurin; IAA = indole-3-acetic acid; 2,4-D = 2,4-dich]orophenoxyaeetic acid; CWP = c e l ] wall polysaccharides; ECP = extra-ce]]ular polysaccharides;

Introduction Plant c e ] l w a l l s do not o n l y have a p r o t e c t i n g and s t r u c t u r a l f u n c t i o n , but a l s o p l a y an important r o l e in morphogenetical and p h y s i o l o g i c a l processes. I t has been shown t h a t the composition of c e l l w a l l s changes during c e l l e ] o n g a t i o n (Franz 1972, Nevins et a l . 1968, N i s h i t a n i and Masuda 1979, VanHo]st e t a ] . 1980), differentiation (Huwyler et a l . 1979, Thornber and Northcote 1962) and m a t u r a t i o n (Yamaki e t a ] . 1979). The c o n t r o l mechanisms of p l a n t c e l l wall b i o s y n t h e s i s connected w i t h p h y s i o l o g i c a l changes of the c e l l are r a t h e r p o o r l y understood. In order to s i m p ] i f y the complex events of growth and d i f f e r e n t i a t i o n in norma] p l a n t s , many i n v e s t i g a t i o n s about the s t r u c t u r e and b i o s y n t h e s i s of c e l l w a l l s have been performed w i t h c e l l c u l t u r e s . Cu]tured c e l l s mostly possess p r i mary c e l l w a l l s . D i f f e r e n t i a t i o n can be induced by the composition of the c u l t u r e media, e s p e c i a l l y by v a r y i n g the c o n c e n t r a t i o n and the combination of phytohormones. However, the c u l t u r e c o n d i t i o n s may a l ready i n f l u e n c e the c e l l wall composition (Takeuchi and Komamine 1978a), and the c e l l wa]] of c u l t u r e d c e ] l s may be d i f f e r e n t from t h a t of t h e i r r e s p e c t i v e mother p l a n t s (Sasaki et a l . 1980).

In t h i s study the sugar composition of l e a f mesop h y l l c e ] l w a l l s From two tobacco v a r i e t i e s is compared to t h a t of r e g e n e r a t i n g p r o t o p l a s t s , c a ] ] i and suspension cultured c e l l s grown in different culture media.

Material and Methods The two tobacco varieties Hicotiana tabacum cv. Xanthi and Hicotiana tabacum cv. Samsun were used. Protoplast isolation and culture of protoplasts, callus and suspension cultured cells have been described by Blaschek et al. (1981). The culture media used were: T-medium = medium according to Nagata and Takebe (1971) with 3 mg/l NAA and ] mg/l 6-BA; S-medium = medium according to Murashige and Skoog (1962) supplemented with 2 mg/] IAA and 0.2 mg/l Kinetin; D-medium = medium according to Murashige and Skoog (1962) supplemented with 0.4 mg/] 2,4-D; M-medium = D-medium supplemented with 0.7 M mannitol. For adaptlon of cells to M-medium the concentration of mannitol was increased in steps of 0.1 - 0.2 M after every 3 - 4 subculturing operations. Cell wall po]ysaecharides (CWP) were prepared from the intercostal regions of leaves (approximat]y 60day o]d plants) and from callus (3 - 4 weeks old); from suspension cultured cells (8 days o]d) or from regenerating protoplasts (3 days old) CWP and extracellular polysaccharides ([CP) were [so]ated according to methods previously described (Blaschek et al.

1981). The analysis of the cell wall sugar composition by gas liquid chromatography of the peracety]ated sugar derivatives was performed according to B]aschek e t a ] . (1981).

Results and Discussion After the analysis of the mesophy11 cell wall of the two tobacco varieties comparable results concerning the neutral sugar composition were obtained. Besides a high content of cel/ulosic glucose of about 50%, arabinose, xylose, galactose and non-cellulosic glucose could be detected as the main polysaccharide constituents. D e t a i l s a r e g i v e n in Fig. I. When protoplasts were isolated from the same mesophyll and cultured in T-medium, a completely different cell wall was regenerated in the time period until the first cell divisions occurd. The CWP of the regener-

258

Fig. I. Neutral sugar c o m p o s i t i o n of cell wall p r e p a r a t i ons from two tobacco varieties.

CWP %[w]

ECP

Rhal~lnose

10

_Arabinose

--

2O I0

IXY lose

I

60 50 40 30 20 I0

80

.

i

70 60 50 40 30 20 10

T T DD

LM F-I= N i c o t i a n a CWP = c e l l

wall

= = = =

C

tabacum cv. X a n t h i ;

nil-

Nicotiana

ECP - e x t r a c e l l u l a r

C = callus;

Nagata/Takebe-medium w i t h Murashige/Skoog's-medium Murashige/Skoog's-medium Murashige/Skoog's-medium

S DD

M M

T

T

SCC

polysaccharides;

LM = l e a f m e s o p h y l l ; T S D M

T TS

TS

S

P

DD

SCC

M M

T

P

tabacum cv. Samsun polysaccharides

SCC - suspension c u l t u r e d

cells;

P = regenerated protoplasts

3 mg/l ,~IAA and I mg/l 6-BA ( 0 . 7 M m a n n i t o l w i t h 2 plg/l IAA and 0.2 mn / l K i n e t i n w i t h 0.4 mg/l 2 . 4 - D w i t h 0.4 mg/l 2 . 4 - D and 0.7 I.i m a n n i t o l

for protoplasts

only)

259 a t i n g p r o t o p l a s t s were composed m a i n l y of n o n - c e l l u losic polysaccharides with glucose predominating (75%) and o n l y minor amounts o f c e l l u l o s e (5%) ( B l a schek et a l . 1981). The c e l l s s e c r e t e d p e c t i c and h e m i c e l l u l o s i c m a t e r i a l i n t o the c u l t u r e medium. This p o l y s a c c h a r i d e f r a c t i o n (ECP) c o n t a i n e d high amounts of g a l a c t o s e and a r a b i n o s e . The unusual c e l l w a l l composition of regenerating protoplasts described also f o r o t h e r p l a n t s p e c i e s (Takeuchi and Komamine 1978b, 1981 and 1982) may be due to a wound response and the u n p h y s i o l o g i e a l c o n d i t i o n s of p r o t o p l a s t i s o l a t i o n and c u l t u r e r e q u i r i n g high o s m o t i c i s o l a t i o n and c u l t u r e media. The development of the c a l l i of both tobacco v a r i e t i e s depended on the c u l t u r e media used. On T-medium w i t h an a u x i n / c y t o k i n i n r a t i o of 3 : 1 , a f i r m , compact, d a r k g r e e n c a l l u s was formed, whereas on a D-medium w i t h a low 2,4-D c o n c e n t r a t i o n as the o n l y p h y t o h o r mone, a l o o s e , f r i a b l e , d i r t y - g r e e n c a l l u s was obt a i n e d . Both c a l l i c o n s i s t e d of u n d i f f e r e n t i a t e d cells. Friability was a s s o c i a t e d w i t h a lower g a ] a c tose c o n t e n t and an a l t e r e d a r a b i n o s e / x y ] o s e r a t i o in the c e l l w a l l s , which was ca. 0.9 f o r compact and ca. 4.3 f o r f r i a b l e c a l l u s . The c e l l w a l l c o m p o s i t i o n o f compact c a l l u s was comparable w i t h t h a t of mesophy]] c e l l s , where a l s o a low a r a b i n o s e / x y l o s e r a t i o of ca. 1.4 c o u l d be d e m o n s t r a t e d . The c o r r e l a t i o n o f changes in the f r i a b i l i t y of calli with altered cell wall c o m p o s i t i o n s has been d e s c r i b e d b e f o r e . In rose suspension c u l t u r e s c e l l u l a r a g g r e g a t e s begin to f r a g ment s p o n t a n e o u s l y a f t e r 12 days o f c u l t u r e , which is accompanied by a decrease of the g a l a c t o s e c o n t e n t of the c e l l w a l l s (Wa]lner and Nevins 1974). F r i a b i l i t y of V i c i a faba c a l l i was accompanied by a decrease o f p e c t i c and h e m i c e ] l u / o s i c m a t e r i a l in the c e l l w a l l s (Grant and F u l / e r 1968). A d i s s o c i a t e d Rosa glauca s t r a i n d i f f e r e d from the compact s t r a i n s by the low p e c t i c p o l y s a c c h a r i d e s c o n t e n t , which showed different r a t i o s o f g a ] a c t o s e and a r a b i n o s e ( M o l ] a r d eta]. 1973). Low k i n e t i n and darkness induced f r i a bility in tobacco c a l l u s , which was c o r r e l a t e d w i t h a g r e a t e r e x t e n s i n c o n t e n t o f the c e l l w a l l and a h i g h e r a r a b i n o s e / x y l o s e r a t i o in the h e m i c e l l u ] o s i c f r a c t i o n (Halmer and Thorpe 1976). High k i n e t i n l e v e l s and darkness led to the f o r m a t i o n o f compact c a l l i . These d i f f e r e n c e s c o u l d not be observed in the l i g h t . These r e s u l t s suggest t h a t the appearence of the t i s s u e t e x t u r e may be dependent on the c o m p o s i t i o n of p e c t i c and h e m i c e l l u l o s i c c e l l w a l l substances i n v o l v e d in the maintenance of i n t e r c e l l u l a r coherency. Changes in c e l l w a l l c o m p o s i t i o n , however, must not be l i n k e d to a change in t i s s u e t e x t u r e and, on the o t h e r hand, v e r y s m a l l , h a r d l y d e t e c t a b l e changes in c e l l w a l l p o l y s a c c h a r i d e s y n t h e s i s may cause a c o m p l e t e l y a l t e r e d p h y s i o l o g i c a l c o n d i t i o n of the c e l l w a l l . Suspension c u l t u r e s were e s t a b l i s h e d in the two media used for the culture of calli and in addition on a S-medium with an auxin/kinetin ratio of 10:1. The cell wall composition in all three media was comparable for both tobacco varieties. Only in S-medium a slightly reduced cellulose content and an increased amount of non-cellulosic glucose was found. In all three culture media an arabinose/xylose ratio of about 3 was detected, which is greater compared to compact calli on T-medium, but lower than in f r i a b l e c a l l u s on D-medium. The suspension c u l t u r e d c e i l s s e c r e t e d s i m i l a r p o ] y s a c c h a r i d e s i n t o the medium, which were composed m a i n l y o f a r a b i n o s e , g a l a c t o s e and g l u c o s e , but c o n t a i n e d a l s o r e l a t i v e l y high amounts of mannose. The ECP o f tobacco c e l l s a r e known to c o n t a i n an a r a b i n o x y l o g l u c a n , a g a l a c t o glucomannan, an ( a r a b i n o ) - x y ] a n and an a r a b i n o g l u curonomannan (Akiyama and Kato 1982). The r e s u l t s suggest t h a t the p h y s i c a l e n v i r o n m e n t of c u l t u r e d p l a n t c e l l s may a l s o i n f l u e n c e the c e l l w a l l composi-

t i o n . I t seems to reduce the c o n t r o l oF c e l l w a l l synt h e s i s by phytohormones. The suspension c u l t u r e d c e l l c l u s t e r s may be o b t a i n e d in a f r i a b l e form due t o a loss o f p o l y s a c c h a r i d e s s o l u b l e in the c u l t u r e medium and due to c o n t i n o u s a g i t a t i o n causing c e l l s e p a r a t i o n . The minor i n f l u e n c e o f phytohormones on the c e l l w a l l c o m p o s i t i o n o f suspension c u l t u r e d c e l l s has a l s o been observed w i t h r e g e n e r a t i n g Finca rosea p r o t o p l a s t s (Takeuchi and Komamine 1982), where p h y t o h o r mones c o u l d c o n t r o l the c e l l d i v i s i o n r a t e , but not the c e l l w a l l c o m p o s i t i o n . P r o t o p ] a s t s , which were c u l t u r e d in high o s m o t i c media, r e g e n e r a t e d an unusual c e l l w a l l . In o r d e r to i n v e s t i g a t e the i n f l u e n c e o f o s m o t i c v a l u e s on c e l l wall b i o s y n t h e s i s , the CWP o f suspension c u l t u r e d c e l l s grown in D-medium were compared to those of c e l l s adapted to M-medium ( = D-medium c o n t a i n i n g 0.7 M m a n n i t o l ) . In f a c t , high o s m o t i c v a l u e s caused an a l t e r e d c e l l w a l l c o m p o s i t i o n . The a r a b i n o s e / x y ] o s e r a t i o was reduced to 1.2 and more mannose and less g a l a c t o s e were found in these c e l l w a l l s . In the ECP more x y l o s e and mannose and less g a ] a c t o s e was present. Hence, the osmolarity of a culture medium is capable to influence the ceil wall composition. The cell walls of regenerating protop]asts, however, were not comparable to those of cells cultured in the same physical environment. In an investigation on regenerating Vinca rosea protoplasts the osmolarity of the culture medium did not affect the sugar composition of the regenerated cell wall (Takeuchi and Komamine 1982), whereas plasmolysis changed the cell wall composition in tobacco leaf discs (Boffey and Northcote 1979). In onion root cells a decrease in the amount of dictyosomes was observed under plasmolysis, which would account for an altered pectin and hemicellu]ose synthesis (Prat 1972). The loss of water from cells cultured in hyperosmotic solutions may change the concentration of ions in the cytoplasm and thus may influence the different po]ysaccharide synthetases. The CWP composition of Nicotiana t a b a c ~ cv. Xanthi and ZVicotiana tabacum cv. Samsun was comparable i n d e pendent o f the c u l t u r e c o n d i t i o n s i n d i c a t i n g a g e n e t ic r e g u l a t i o n of the b i o s y n t h e s i s of the c e l l w a l l . This is confirmed by the o b s e r v a t i o n s w i t h o t h e r p l a n t s p e c i e s , where the v a r i e t i e s w i t h i n a g i v e n s p e c i e s had i d e n t i c a l , but d i f f e r e n t species of the same or of d i f f e r e n t genera had d i f f e r e n t c e l l w a l l c o m p o s i t i o n s (Nevins e t a ] . 1967). One can c o n c l u d e t h a t p l a n t c e l l s s y n t h e s i z e an a l t e r e d c e l t w a l l not o n l y d u r i n g c e l l growth and d i f f e r entiation, but a l s o under the i n f l u e n c e o f e x t e r n a l f a c t o r s l i k e phytohormones (Ha]mer and Thorpe 1976, N i s h i t a n i and Masuda 1981, Sakurai e t a ] . 1979), carbon sources (Nevins et a l . 1967), ion c o m p o s i t i o n of the c u ] t u r e media (Dugger and Palmer 1980), o s m o t i c v a l u e s , l i g h t (Ha]met and Thorpe 1976) and the p h y s i c a l e n v i r o n m e n t . A l l these f a c t o r s should be cons i d e r e d in i n v e s t i g a t i o n s c o n c e r n i n g the c e l l w a l l b i o s y n t h e s i s in c u l t u r e d p l a n t c e l l s .

Acknowledgements This work was supported by the Deutsche Forschungsgem e i n s c h a f t ( S o n d e r f o r s c h u n g s b e r e i c h 4 3 / A 1 ) . The aut h o r s wish to thank Miss Uschi Semler and Miss Gabi Stadler for skilful technical assistance.

260 References Akiyama Y, Kato K (1982) Phytochem 2 1 : 1 3 2 5

- 1329

B1aschek W, Haass D, Koehler H, Franz G (1981) Plant Sci Lett 2 2 : 4 7 - 57 Boffey SA, Northcote 433 - 440

DH (1975) Biochem J 150:

- 347

Ha]met P, Thorpe TA (1976) Phytochem 1585 - 1588

- 680

21

Mollard A, Hustache G, Barnoud F (1973) Physiol V~g 1 1 : 5 3 9 - 552 Plant 15:

I (1970) P]anta 9 2 : 3 0 1

Nevins DJ, English PD, Albersheim Plant Physiol 4 2 : 9 0 0 - 906

- 308

P (1967)

Nevins DJ, English PD, Albersheim P (1968) Plant Physio] 4 3 : 9 1 4 - 922

P]ant 52:

14:85

- 114

Sakurai N, Nishitani K, Masuda Y (1979) Plant Cell Physiol 2 0 : 1 3 4 9 - 1357 Takeuchi

15:

Huwyler HR, Franz G, Meier H (1979) Planta 146: 635 - 642

Murashige T, Skoog F (1962) Physio] 473 - 479

(Paris)

20:

Sasaki K, Maeda M, Ohira K (1980) Plant Cell Physiol 2 1 : 2 6 5 - 278

65:

Grant ME, Fuller KW (1968) J Exp Bot 1 9 : 6 6 7

Nagata T, Takebe

Nishitani K, Masuda Y (1981) Physiol 482 - 494 Prat (1972) J Microscop

Dugger WM, Palmer RL (1980) Plant Physiol 266 - 273 Franz G (1972) Planta 1 0 2 : 3 3 4

Nishitani K, Masuda Y (1979) Plant Cell Physiol 63 - 74

-

Y, Komamine A (1978a) Physiol

Plant 42:

28

Takeuchi

Y, Komamine A (1978b) Planta 1 4 0 : 2 2 7

- 232

Takeuchi Y, Komamine A (1981) Plant Cell Physio] 1585 - 1594

22:

Takeuchi Y, Komamine A (1982) Plant Cell Physio] 249 - 255

23:

Thornber JP, Northcote DH (1962) Biochem J 82: 340 - 346 VanHolst GJ, Klis FM, Bouman F, Stegwee D (1980) P1anta 1 4 9 : 2 0 9 - 212 Wallner SJ, Nevins DJ (1974) J Exp Bot 25: 1020 - 1029 Yamaki S, Machida Y, Kakiuchi N (1979) Plant Cell Physiol 2 0 : 3 1 1 - 321