Plant Cell Reports (1983) 2:292-295
Plant Cell Reports © Springer-Verlag 1983
Organelles Associated with the Plasma Membrane of Tobacco Leaf Protoplasts L. C. F o w k e 1, p. j. R e n n i e 1, a n d F. C o n s t a b e l 2 1 Department of Biology, University of Saskatchewan, Saskatoon, Sask., Canada, S7N 0W0 2 Plant Biotechnology Institute, National Research Council, Saskatoon, Sask., Canada, S7N 0W9 Received September 12, 1983 / October 7, 1983 Communicated by J. K. Vasil
ABSTRACT
MATERIALS
Leaf p r o t o p l a s t s of t o b a c c o ( N i c o t i a n a t a b a cum) h e t e r o z y g o u s (Su/su) and n o r m a l (su/su) for the s u l f u r m u t a t i o n e x h i b i t a c h a r a c t e r i s t i c lag p e r i o d b e f o r e i n i t i a t i n g c e l l w a l l formation. The e a r l y w a l l is c o m p o s e d of a n e t w o r k of C a l c o f l u o r p o s i t i v e f i b r i l s . L a r g e f r a g m e n t s of p l a s m a m e m b r a n e f r o m f r e s h l y isolated protoplasts were examined by elect r o n m i c r o s c o p y to d e t e r m i n e the d i s t r i b u t i o n of a s s o c i a t e d o r g a n e l l e s . C o a t e d v e s i c l e s and p a t c h e s of c o a t m a t e r i a l w e r e p r e s e n t on the i n n e r s u r f a c e of the p l a s m a m e m b r a n e . T h e f r e q u e n c y of c o a t e d v e s i c l e s o b s e r v e d w a s c o n s i d e r a b l y less than r e p o r t e d for p r o t o plasts from suspension cultured tobacco cells. V e r y few m i c r o t u b u l e s w e r e a s s o c i a t e d w i t h the p l a s m a m e m b r a n e b u t fine f i l a m e n t s were frequently observed.
Plant
INTRODUCTION
P r o t o p l a s t s p r o v i d e a u s e f u l tool for i n v e s t i g a t i n g the s t r u c t u r e and f u n c t i o n of p l a n t c e l l o r g a n e l l e s (see r e v i e w s b y F o w k e and G a m b o r g 1980, F o w k e and C o n s t a b e l 1982). P r o t o p l a s t s are p a r t i c u l a r l y u s e f u l for i s o l a ting and c h a r a c t e r i z i n g a v a r i e t y of o r g a n e l l e s (e.g. v a c u o l e s , p l a s t i d s , m i t o c h o n d r i a , chromosomes, nuclei, coated vesicles). The r e m o v a l of the c e l l w a l l f r o m p l a n t c e l l s also p e r m i t s the s t u d y of the p l a s m a m e m brane. The p l a n t p l a s m a m e m b r a n e r e g u l a t e s t r a n s p o r t b e t w e e n a p o p l a s t and s y m p l a s t and p a r t i c i p a t e s in the d e p o s i t i o n of the c e l l w a l l ( L e o n a r d and H o d g e s 1980, F o w k e 1983). M a r c h a n t (1978) d e v e l o p e d an i n t e r e s t i n g t e c h n i q u e for e x a m i n i n g the i n n e r s u r f a c e of a l g a l p l a s m a m e m b r a n e s and s t u d y i n g the ass o c i a t e d o r g a n e l l e s w h i c h m a y b e i n v o l v e d in t h e s e i m p o r t a n t p r o c e s s e s . This m e t h o d has b e e n s u c c e s s f u l l y a p p l i e d to h i g h e r p l a n t p r o t o p l a s t s ( D o o h a n and P a l e v i t z 1980, L l o y d et al. 1980, Van d e r V a l k et al. 1980). Inf o r m a t i o n r e g a r d i n g the s t r u c t u r e and d i s t r i h u t i o n of o r g a n e l l e s on the p l a s m a m e m b r a n e s of p r o t o p l a s t s c a p a b l e of r a p i d w a l l r e g e n e r a t i o n has b e e n p r e v i o u s l y r e p o r t e d (Van d e r V a l k et al. 1980, Van d e r V a l k a n d F o w k e 1981). T h i s s t u d y e x a m i n e s the p l a s m a m e m b r a n e s of leaf p r o t o p l a s t s w h i c h u n d e r g o a lag p h a s e b e f o r e i n i t i a t i n g c e l l w a l l d e p o s i t i o n ( F o w k e and G a m b o r g 1980, W i l l i s o n and K l e i n 1982).
AND
METHODS
Material
Seeds from Nicotiana tabacum heterozygous for the s u l f u r m u t a t i o n (Su/su) w e r e g e r m i nated aseptically on a g a r c o n t a i n i n g n u t r i e n t s (OMS) a c c o r d i n g to M u r a s h i g e and S k o o g (1962). H e t e r o z y g o u s (Su/su) and n o r m a l (su/su) s e e d l i n g s w e r e s e l e c t e d a n d t r a n s f e r r e d to 'peat pots' for s e v e r a l w e e k s and t h e n to c l a y f l o w e r p o t s in a i : i : i sand: peat:soil mixture. Plants were maintained u n d e r b o t h g r e e n h o u s e c o n d i t i o n s and in c o n t r o l l e d e n v i r o n m e n t c h a m b e r s w i t h l i g h t int e n s i t i e s of 2 0 0 0 - 3 8 0 0 lux. T h e y w e r e f e r t i l i z e d p e r i o d i c a l l y w i t h 2x H o a g l a n d ' s n u t r i ent s o l u t i o n . Protoplast
Isolation
F u l l y e x p a n d e d y o u n g l e a v e s of n o n - f l o w e r i n g p l a n t s w e r e e x c i s e d , r i n s e d in 70% e t h a n o l for 25 sec, s u b m e r g e d for i0 m i n in 1 . 2 % s o d i u m h y p o c h l o r i t e and r i n s e d in s t e r i l e w a t e r . The l o w e r e p i d e r m i s ~ a s r e m o v e d u n d e r aseptic c o n d i t i o n s . One cm p l e c e s of leaf w e r e f l o a t e d on the f o l l o w i n g e n z y m e s o l u t i o n at r o o m t e m p e r a t u r e on a g y r a t o r y s h a k e r (30 rpm) for 4 hr: 1 . 5 % (w/v) c e l l u l a s e (Onozuka R-10), 0 . 5 % (w/v) p e c t i n a s e in 0. 7M sorb i t o l w i t h 2 m M CaH. ( P O . ) ~ . H . O at pH 5.8. z z P r o t o p l a s t s w e r e f i l t e r e d t h r o u g h a 50 ~m nylon mesh and washed three times with wash solution (0.7M sorbitol containing 2 mM CaH4(PO4)2"H20)" Protoplast
Culture
for Wall
Regeneration
W a s h e d p r o t o p l a s t s w e r e c u l t u r e d in s m a l l d r o p l e t s of c u l t u r e m e d i u m (Kao 1982) in petri dishes sealed with parafilm and incub a t e d at r o o m t e m p e r a t u r e in h u m i d i f i e d p l a s t i c c o n t a i n e r s in s u b d u e d light. S a m p l e s w e r e t a k e n for l i g h t m i c r o s c o p y at 0 time, 2, 4, 6, 8 and 24 h o u r s and 5 days. P r o t o plasts were stained with 0.1% Calcofluor W h i t e M2R New ( A m e r i c a n C y a n a m i d ) in 0.7 M s o r b i t o l for 2-5 m i n at r o o m t e m p e r a t u r e and e x a m i n e d b y f l u o r e s c e n c e m i c r o s c o p y to m o n i tor c e l l w a l l r e g e n e r a t i o n . Membrane Washed lysine
Preparations
for
protoplasts were t r e a t e d 200 m e s h
Electron
Microscopy
a t t a c h e d to p o l y formvar/carbon coated
293 grids. They were carefully r i n s e d in t h e w a s h s o l u t i o n a n d t h e n s u b m e r g e d in a l a r g e v o l u m e of m i c r o t u b u l e stabilizing buffer (MSB - i00 mM PIPES buffer 1.0 m M MgSO4, 2.0 m M EGTA, pH a d j u s t e d to 6.9) f o r 20 m l n to o s m o t i c a l l y r u p t u r e the p r o t o p l a s t s . The plasma membrane fragments a d h e r i n g to the g r i d s w e r e w a s h e d 3 t i m e s w i t h MSB, f i x e d in 2% g l u t a r a l d e h y d e in M S B f o r i0 min, r i n s e d w e l l w i t h M S B a n d air dried. The membrane fragments were then s t a i n e d w i t h 2% a q u e o u s u r a n y l a c e t a t e a n d e x a m i n e d in a P h i l i p s 300 e l e c t r o n m i c r o : scope. P h o t o g r a p h i c p r i n t s of r a n d o m l y sel e c t e d r e g i o n s of m e m b r a n e w e r e a n a l y z e d u s i n g s t a n d a r d a r e a s to d e t e r m i n e the freq u e n c y of c o a t e d v e s i c l e s . F i g u r e 5 h a s b e e n i n c l u d e d in t h i s p a p e r to permit a visual comparison of p l a s m a m e m branes from protoplasts of s u s p e n s i o n cultured cells and leaf protoplasts. Details
r e g a r d i n g the p r e p a r a t i o n of t h e m e m b r a n e illustrated in F i g u r e 5 as w e l l as i n f o r m a tion regarding coated vesicle frequency have been previously reported (Van d e r V a l k et al. 1980, Van d e r V a l k a n d F o w k e 1981). T h e c e l l suspension used by Van der Valk and co-workers is g e n e t i c a l l y s i m i l a r to the l e a f m a t e r i a l u s e d in the c u r r e n t s t u d y s i n c e it w a s originally d e r i v e d f r o m t i s s u e of a l b i n o seedlings (Su/su). P r o t o p l a s t s from albino leaves could not be obtained for this study b e c a u s e of t e c h n i c a l d i f f i c u l t i e s . RESULTS
AND
DISCUSSION
Cultured leaf protoplasts exhibited a charact e r i s t i c lag p e r i o d b e f o r e r e g e n e r a t i n g suff i c i e n t c e l l w a l l to b e d e t e c t e d b y C a l c o fluor staining (see F o w k e a n d G a m b o r g 1980, W i l l i s o n a n d K l e i n 1982). A f t e r 4 hr c u l t u r e most protoplasts w e r e d e v o i d of d e t e c t a b l e
Fig. 1 Light micrograph showing Calcofluor p o s i t i v e f i b r i l s on t h e s u r f a c e of t o b a o c o l e a f protoplasts c u l t u r e d f o r 8 hr. b a r : 30 ~m. Fig. 2 Electron micrograph showing a negatively s t a i n e d f r a g m e n t of t o b a c c o l e a f p l a s m a m e m b r a n e . Coated vesicles (double arrows), fine filam e n t s ( s i n g l e a r r o w s ) and a m i c r o t u b u l e (Mt) are p r e s e n t , b a r : 200 nm. Fig. 3 Electron micrograph of a n e g a t i v e l y s t a i n e d f r a g m e n t of t o b a c c o l e a f p l a s m a m e m b r a n e s h o w i n g a p a t c h of a l v e o l a t e c o a t m a t e r i a l , b a r = 200 nm. Fig. 4 Electron micrograph of a f r a g m e n t of t o b a c c o leaf p l a s m a m e m b r a n e s h o w i n g d i s t r i b u t i o n of c o a t e d v e s i c l e s (arrows).bar = 0.5 ~m. Fig. 5 Electron micrograph of a f r a g m e n t of p l a s m a m e m b r a n e f r o m s u s p e n s i o n c u l t u r e d t o b a c c o cell showing distribution of c o a t e d v e s i c l e s (single arrows). N o t e the b u n d l e of m i c r o t u b u l e s ( d o u b l e a r r o w ) , b a r : 0.5 ~m ( k i n d l y s u p p l i e d by P. V a n d e r Valk).
294 Calcofluor s t a i n i n g b u t b y 6 hr p r o t o p l a s t s were enclosed within a faintly staining fibr i l l a r n e t w o r k . By 8 hr an e x t e n s i v e n e t w o r k of C a l c o f l u o r - p o s i t i v e fibrils was observed on the m a j o r i t y of p r o t o p l a s t s (Fig. i). A f t e r 5 d a y s in c u l t u r e the f i b r i l l a r n e t w o r k h a d b e e n r e p l a c e d by a u n i f o r m l y fluores c e n t c e l l wall. T h e n a t u r e of t h e f i b r i l s on the c u l t u r e d l e a f p r o t o p l a s t s has n o t as y e t been established. Calcofluor a l o n e is n o t a r e l i a b l e i n d i c a t o r for c e l l u l o s e b e c a u s e it can s t a i n o t h e r p o l y s a c c h a r i d e s (see W i l l i s o n and K l e i n 1982). F i b r i l s h a v e b e e n d e m o n s t r a t e d on the s u r f a c e of c u l t u r e d p r o t o p l a s t s b y a v a r i e t y of t e c h n i q u e s (e.g., n e g a t i v e staining, freeze-etching, replicas) and recent biochemical evidence suggests that they a r e c o m p o s e d of c e l l u l o s e (e.g., K l e i n et al. 1981). C o a t e d v e s i c l e s w e r e o b s e r v e d on the i n n e r s u r f a c e of the p l a s m a m e m b r a n e of b o t h S u / s u heterozygous and n o r m a l p l a n t s . T h e i r s i z e a n d s t r u c t u r e a p p e a r e d s i m i l a r to t h a t r e p o r t e d f o r c o a t e d v e s i c l e s on p l a s m a m e m branes from suspension cultured tobacco cells (Fig. 2 cf. V a n d e r V a l k a n d F o w k e 1981). It is i m p o r t a n t to r e a l i z e t h a t a n u m b e r of the structures identified in t h i s p a p e r as c o a t e d vesicles, are in f a c t c o a t e d p i t s ( v e s i c l e s incorporated i n t o the p l a s m a m e m b r a n e ) . However, it is n o t p o s s i b l e to d i s t i n g u i s h t h e s e two s t r u c t u r e s in s u r f a c e v i e w s of the plasma membrane. Occasionally large regions of a l v e o l a t e c o a t w e r e o b s e r v e d on t h e p l a s m a membrane (Fig. 3). S i m i l a r s t r u c t u r e s h a v e been reported previously for both plant and animal plasma membranes (Van d e r V a l k a n d F o w k e 1981, A g g e l e r a n d W e r b 1982, M a u p i n a n d P o l l a r d 1983) b u t t h e i r s i g n i f i c a n c e is unknown. R e s u l t s of t h r e e s e p a r a t e e x p e r i m e n t s revealed average frequencies ~f 1.54, 0 . 6 6 and 0 . 6 7 c o a t e d v e s i c l e s p e r Bm of p l a s m a m e m b r a n e ( T a b l e i). The n u m b e r of c o a t e d 2 v e s i c l e s p e r ~m r a n g e d f r o m 0 . 2 9 to 2.70 o v e r the t h r e e e x p e r i m e n t s . The d i f f e r e n c e in the a v e r a g e n u m b e r of c o a t e d v e s i c l e s o b served between experiment 1 and the o t h e r two e x p e r i m e n t s m a y b e d u e to v a r i a t i o n s in the age of l e a v e s used. T h e l e a v e s f o r experiment 1 were approximately 6-8 cm in l e n g t h w h i l e t h o s e f o r the o t h e r e x p e r i m e n t s e x c e e d 13 cm in l e n g t h . D e s p i t e the v a r i a b i -
lity observed, it is c l e a r t h a t the f r e q u e n c y of c o a t e d v e s i c l e s On l e a f p l a s m a m e m b r a n e s is c o n s i d e r a b l y l o w e r t h a n on p l a s m a m e m b r a n e s of p r o t o p l a s t s derived from suspension c u l t u r e d t o b a c c o c e l l s (SU 677). V a n d e r V a l k a n d F o w k e (1981) r e p o r t e d an a v e r a g e of 8.0 c o a t e d v e s i c l e s p e r ~m of m e m b r a n e w i t h a r a n g e of 3-12 p e r ~m of m e m b r a n e . Figure 4 and 5 illustrate the d i f f e r e n c e in c o a t e d v e s i c l e f r e q u e n c y b e t w e e n the two t y p e s of p r o t o plasts. The significance of t h i s d i f f e r e n c e is u n c l e a r at p r e s e n t . C o a t e d v e s i c l e s m a y h a v e "a r o l e in p l a n t c e l l w a l l d e p o s i t i o n (see N e w c o m b 1980) a n d t h e y are o f t e n l o c a l i z e d in r e g i o n s of a c t i v e c e l l w a l l f o r m a t i o n s u c h as f o r m i n g c e l l p l a t e s ( F r a n k e a n d H e r t h 1974, N a k a m u r a a n d M i k i - H i r o s h i g e 1982) a n d the t i p s of a c t i v e l y g r o w i n g r o o t h a i r s ( R o b e r t s o n a n d L y t t l e t o n 1982) a n d c o t t o n h a i r s ( R y s e r 1979). If c o a t e d v e s i c l e s h a v e a r o l e in t r a n s p o r t i n g m a t e r i a l s to the r e generating cell wall, cell culture protop l a s t s w h i c h a r e c a p a b l e of r a p i d i n i t i a t i o n of n e w w a l l w o u l d be e x p e c t e d to h a v e m o r e coated vesiclesassociated with their plasma membrane than leaf protoplasts which experience a pronounced lag p r i o r to w a l l f o r m a tion. C e l l w a l l f o r m a t i o n in P l a n t s i n v o l v e s t h e t r a n s p o r t of m a t r i x m a t e r i a l s to the c e l l surface via Golgi vesicles which fuse with and contribute to the p l a s m a m e m b r a n e . It is conceivable that coated vesicles might funct i o n by r e c y c l i n g p l a s m a m e m b r a n e b a c k i n t o the c y t o p l a s m . A h i g h e r f r e q u e n c y of c o a t e d v e s i c l e s on p l a s m a m e m b r a n e s of s u s p e n s i o n culture protoplasts would be consistent with t h i s i d e a as w e l l . Recent evidence suggests that coated vesicles m i g h t h a v e a r o l e in the p r o c e s s of e n d o c y t o s i s by p l a n t s ( T a n c h a k et al. 1 9 8 3 ) . T h e difference in c o a t e d v e s i c l e f r e q u e n c i e s obs e r v e d in the p r e s e n t s t u d y m i g h t t h e r e f o r e reflect general differences in m e t a b o l i c a c t i v i t i e s of p r o t o p l a s t s from rapidly growing suspension cultured cells and mature leaf cells. very few microtubules w e r e o b s e r v e d on l e a f plasma membranes (Fig. 2, 4). In c o n t r a s t , plasma membrane fragments from protoplasts c a p a b l e of a c t i v e w a l l s e c r e t i o n a r e c h a r a c terized by numerous microtubules (Van d e r V a l k et al. 1980). T h e d i r e c t c o r r e l a t i o n
T a b l e i. Frequency Leaf Protoplasts
Membrane
of
Coated
Vesicles
Number Experiment 1
2
3
aRange
Fragments
from
Nicotiana
Examined
T o t a l A r e a o~ Membrane (~m -)
A v e r a g e N u m b e r of Coated Vesicles per
Heterozygous p l a n t s (Su/su)
ii
i158
1.54
(0.44
- 2.70) a
Heterozygous p l a n t s (Su/su)
9
808
0.66
(0.35
- 1.21)
15
1060
0.67
(0.29
- 1.14)
Normal plants
given
Plasma
(su/su)
in p a r e n t h e s e s .
tabacum
of
Protoplasts
Leaf Source
on
~m
2
295 b e t w e e n p r e s e n c e of m i c r o t u b u l e s on the p l a s m a m e m b r a n e and a b i l i t y to s y n t h e s i z e c e l l w a l l is p e r h a p s not s u r p r i s i n g s i n c e m i c r o t u b u l e s s e e m to h a v e an i m p o r t a n t r o l e in o r i e n t i n g c e l l u l o s e m i c r o f i b r i l d e p o s i t i o n ( r e v i e w e d by G u n n i n g and H a r d h a m 1982 Fowke et al. 1983). F i n e f i l a m e n t s w e r e o f t e n o b s e r v e d on f r a g m e n t s of leaf p l a s m a m e m b r a n e (Fig. 2 The n a t u r e of t h e s e f i l a m e n t s is p r e s e n t l y u n k n o w n . It is p o s s i b l e that t h e y are c o m p o s e d of a c t i n s i n c e a c t i n m i c r o f i l a m e n t s h a v e b e e n i d e n t i f i e d in b o t h a l g a l and h i g h e r p l a n t p r o t o p l a s t s ( M a r c h a n t 1976, Y a m a g u c h i and N a g a i 1981). A l t e r n a t i v e l y , t h e y m a y not be c o m p o s e d of a c t i n and m a y r e p r e s e n t p a r t of a f i l a m e n t o u s n e t w o r k b e l i e v e d to p r o v i d e s u p p o r t f o r the n u c l e u s in p l a n t p r o t o p l a s t s ( P o w e l l et al. 1982).
ACKNOWLEDGEMENTS
The a s s i s t a n c e of Mr. J. S h y l u k is g r e a t l y appreciated. This project was supported by the N a t u r a l S c i e n c e s a n d E n g i n e e r i n g R e s e a r c h C o u n c i l of C a n a d a .
REFERENCES
A g g e l e r J, W e r b Z (1982) J C e l l B i o l 95: 613-623 D o o h a n ME, P a l e v i t z BA (1980) P l a n t a 149: 389-401 F o w k e LC (1983) In: G a r r o d D, C h a d w i c k C (eds) R e c e p t o r s in p l a n t s and s l i m e m o u l d s , M a r c e l D e k k e r , New York, (in press) F o w k e LC, C o n s t a b l e F (1982) In: G i l e s KL, Sen SK (eds) P l a n t c e l l c u l t u r e in c r o p improvement, Plenum Publishing Corporation, N e w York, pp 1 8 5 - 2 0 0 F o w k e LC, G a m b o r g OL (1980) Int Rev C y t o l 68: 9-51
F o w k e LC, G r i l l i n g LR, M e r s e y BG, Van d e r V a l k P (1983) P r o c e e d 6th Int P r o t o p l a s t Symp, B a s e l S w i t z e r l a n d F r a n k e WW, H e r t h W (1974) E x p t l C e l l Res 89: 447-451 G u n n i n g BES, H a r d h a m AR (1982) Ann Rev P l a n t P h y s i o l 33: 6 5 1 - 6 9 8 Kao KN (1982) In: W e t t e r LR, C o n s t a b e l F (eds) P l a n t t i s s u e c u l t u r e m e t h o d s , N a t i o nal R e s e a r c h C o u n c i l of C a n a d a , S a s k a t o o n , pp 49-56 K l e i n AS, M o n t e z i n o s D, D e l m e r DP (1981) P l a n t a 152: 1 0 5 - 1 1 4 L e o n a r d RT, H o d g e s T K (1980) In: T o l b e r t NE (ed) T h e b i o c h e m i s t r y of p l a n t s , A c a d e m i c Press, New York, pp 1 6 3 - 1 8 2 L l o y d CW, S l a b a s AR, P o w e l l AJ, L o w e SB (1980) P l a n t a 147: 5 0 0 - 5 0 6 M a r c h a n t HJ (1976) P l a n t a l 3 1 : 1 1 9 - 1 2 0 M a r c h a n t HJ (1978) E x p t l C e l l Res 115: 2 5 - 3 0 M a u p i n P, P o l l a r d TD (1983) J C e l l B i o l 96: 51-62 M u r a s h i g e T, S k o o g F (1962) P h y s i o l P l a n t 15: 431-497 N a k a m u r a S, M i k i - H i r o s h i g e H (1982) J U l t r a s t r u c Res 80: 3 0 2 - 3 1 1 N e w c o m b EH (1982) In: O c k l e f o r d CJ, W h y t e A (eds) C o a t e d v e s i c l e s , C a m b r i d g e U n i v e r s i t y Press, New York, pp 5 5 - 6 8 P o w e l l AJ, P e a c e GW, S l a b a s AR, L l o y d C W (1982) J C e l l Sci 56: 3 1 9 - 3 3 5 R o b e r t s o n JG, L y t t l e t o n P (1982) J C e l l Sci 58: 6 3 - 7 8 R y s e r U (1979) P r o t o p l a s m a 98: 2 2 3 - 2 3 9 T a n c h a k M, G r i f f i n g LR, M e r s e y BG, F o w k e LC (1983) J C e l l B i o l ( A b s t r a c t ) Van d e r V a l k P, F o w k e LC (1981) C a n J Bot 59: 1307-1313 Van d e r V a l k P, R e n n i e PJ, C o n n o l l y JA, F o w k e LC (1980) P r o t o p l a s m a 105: 2 7 - 4 3 W i l l i s o n JHM, K l e i n AS (1982) In: B r o w n R M (ed) C e l l u l o s e and o t h e r n a t u r a l p o l y m e r s y s t e m s , P l e n u m , N e w Y o r k , pp 6 1 - 8 5 Y a m a g u c h i Y, N a g a i R (1981) J C e l l Sci 48: 193-205
Plant Cell Reports © Springer-Verlag 1983
Organelles Associated with the Plasma Membrane of Tobacco Leaf Protoplasts L. C. F o w k e 1, p. j. R e n n i e 1, a n d F. C o n s t a b e l 2 1 Department of Biology, University of Saskatchewan, Saskatoon, Sask., Canada, S7N 0W0 2 Plant Biotechnology Institute, National Research Council, Saskatoon, Sask., Canada, S7N 0W9 Received September 12, 1983 / October 7, 1983 Communicated by J. K. Vasil
ABSTRACT
MATERIALS
Leaf p r o t o p l a s t s of t o b a c c o ( N i c o t i a n a t a b a cum) h e t e r o z y g o u s (Su/su) and n o r m a l (su/su) for the s u l f u r m u t a t i o n e x h i b i t a c h a r a c t e r i s t i c lag p e r i o d b e f o r e i n i t i a t i n g c e l l w a l l formation. The e a r l y w a l l is c o m p o s e d of a n e t w o r k of C a l c o f l u o r p o s i t i v e f i b r i l s . L a r g e f r a g m e n t s of p l a s m a m e m b r a n e f r o m f r e s h l y isolated protoplasts were examined by elect r o n m i c r o s c o p y to d e t e r m i n e the d i s t r i b u t i o n of a s s o c i a t e d o r g a n e l l e s . C o a t e d v e s i c l e s and p a t c h e s of c o a t m a t e r i a l w e r e p r e s e n t on the i n n e r s u r f a c e of the p l a s m a m e m b r a n e . T h e f r e q u e n c y of c o a t e d v e s i c l e s o b s e r v e d w a s c o n s i d e r a b l y less than r e p o r t e d for p r o t o plasts from suspension cultured tobacco cells. V e r y few m i c r o t u b u l e s w e r e a s s o c i a t e d w i t h the p l a s m a m e m b r a n e b u t fine f i l a m e n t s were frequently observed.
Plant
INTRODUCTION
P r o t o p l a s t s p r o v i d e a u s e f u l tool for i n v e s t i g a t i n g the s t r u c t u r e and f u n c t i o n of p l a n t c e l l o r g a n e l l e s (see r e v i e w s b y F o w k e and G a m b o r g 1980, F o w k e and C o n s t a b e l 1982). P r o t o p l a s t s are p a r t i c u l a r l y u s e f u l for i s o l a ting and c h a r a c t e r i z i n g a v a r i e t y of o r g a n e l l e s (e.g. v a c u o l e s , p l a s t i d s , m i t o c h o n d r i a , chromosomes, nuclei, coated vesicles). The r e m o v a l of the c e l l w a l l f r o m p l a n t c e l l s also p e r m i t s the s t u d y of the p l a s m a m e m brane. The p l a n t p l a s m a m e m b r a n e r e g u l a t e s t r a n s p o r t b e t w e e n a p o p l a s t and s y m p l a s t and p a r t i c i p a t e s in the d e p o s i t i o n of the c e l l w a l l ( L e o n a r d and H o d g e s 1980, F o w k e 1983). M a r c h a n t (1978) d e v e l o p e d an i n t e r e s t i n g t e c h n i q u e for e x a m i n i n g the i n n e r s u r f a c e of a l g a l p l a s m a m e m b r a n e s and s t u d y i n g the ass o c i a t e d o r g a n e l l e s w h i c h m a y b e i n v o l v e d in t h e s e i m p o r t a n t p r o c e s s e s . This m e t h o d has b e e n s u c c e s s f u l l y a p p l i e d to h i g h e r p l a n t p r o t o p l a s t s ( D o o h a n and P a l e v i t z 1980, L l o y d et al. 1980, Van d e r V a l k et al. 1980). Inf o r m a t i o n r e g a r d i n g the s t r u c t u r e and d i s t r i h u t i o n of o r g a n e l l e s on the p l a s m a m e m b r a n e s of p r o t o p l a s t s c a p a b l e of r a p i d w a l l r e g e n e r a t i o n has b e e n p r e v i o u s l y r e p o r t e d (Van d e r V a l k et al. 1980, Van d e r V a l k a n d F o w k e 1981). T h i s s t u d y e x a m i n e s the p l a s m a m e m b r a n e s of leaf p r o t o p l a s t s w h i c h u n d e r g o a lag p h a s e b e f o r e i n i t i a t i n g c e l l w a l l d e p o s i t i o n ( F o w k e and G a m b o r g 1980, W i l l i s o n and K l e i n 1982).
AND
METHODS
Material
Seeds from Nicotiana tabacum heterozygous for the s u l f u r m u t a t i o n (Su/su) w e r e g e r m i nated aseptically on a g a r c o n t a i n i n g n u t r i e n t s (OMS) a c c o r d i n g to M u r a s h i g e and S k o o g (1962). H e t e r o z y g o u s (Su/su) and n o r m a l (su/su) s e e d l i n g s w e r e s e l e c t e d a n d t r a n s f e r r e d to 'peat pots' for s e v e r a l w e e k s and t h e n to c l a y f l o w e r p o t s in a i : i : i sand: peat:soil mixture. Plants were maintained u n d e r b o t h g r e e n h o u s e c o n d i t i o n s and in c o n t r o l l e d e n v i r o n m e n t c h a m b e r s w i t h l i g h t int e n s i t i e s of 2 0 0 0 - 3 8 0 0 lux. T h e y w e r e f e r t i l i z e d p e r i o d i c a l l y w i t h 2x H o a g l a n d ' s n u t r i ent s o l u t i o n . Protoplast
Isolation
F u l l y e x p a n d e d y o u n g l e a v e s of n o n - f l o w e r i n g p l a n t s w e r e e x c i s e d , r i n s e d in 70% e t h a n o l for 25 sec, s u b m e r g e d for i0 m i n in 1 . 2 % s o d i u m h y p o c h l o r i t e and r i n s e d in s t e r i l e w a t e r . The l o w e r e p i d e r m i s ~ a s r e m o v e d u n d e r aseptic c o n d i t i o n s . One cm p l e c e s of leaf w e r e f l o a t e d on the f o l l o w i n g e n z y m e s o l u t i o n at r o o m t e m p e r a t u r e on a g y r a t o r y s h a k e r (30 rpm) for 4 hr: 1 . 5 % (w/v) c e l l u l a s e (Onozuka R-10), 0 . 5 % (w/v) p e c t i n a s e in 0. 7M sorb i t o l w i t h 2 m M CaH. ( P O . ) ~ . H . O at pH 5.8. z z P r o t o p l a s t s w e r e f i l t e r e d t h r o u g h a 50 ~m nylon mesh and washed three times with wash solution (0.7M sorbitol containing 2 mM CaH4(PO4)2"H20)" Protoplast
Culture
for Wall
Regeneration
W a s h e d p r o t o p l a s t s w e r e c u l t u r e d in s m a l l d r o p l e t s of c u l t u r e m e d i u m (Kao 1982) in petri dishes sealed with parafilm and incub a t e d at r o o m t e m p e r a t u r e in h u m i d i f i e d p l a s t i c c o n t a i n e r s in s u b d u e d light. S a m p l e s w e r e t a k e n for l i g h t m i c r o s c o p y at 0 time, 2, 4, 6, 8 and 24 h o u r s and 5 days. P r o t o plasts were stained with 0.1% Calcofluor W h i t e M2R New ( A m e r i c a n C y a n a m i d ) in 0.7 M s o r b i t o l for 2-5 m i n at r o o m t e m p e r a t u r e and e x a m i n e d b y f l u o r e s c e n c e m i c r o s c o p y to m o n i tor c e l l w a l l r e g e n e r a t i o n . Membrane Washed lysine
Preparations
for
protoplasts were t r e a t e d 200 m e s h
Electron
Microscopy
a t t a c h e d to p o l y formvar/carbon coated
293 grids. They were carefully r i n s e d in t h e w a s h s o l u t i o n a n d t h e n s u b m e r g e d in a l a r g e v o l u m e of m i c r o t u b u l e stabilizing buffer (MSB - i00 mM PIPES buffer 1.0 m M MgSO4, 2.0 m M EGTA, pH a d j u s t e d to 6.9) f o r 20 m l n to o s m o t i c a l l y r u p t u r e the p r o t o p l a s t s . The plasma membrane fragments a d h e r i n g to the g r i d s w e r e w a s h e d 3 t i m e s w i t h MSB, f i x e d in 2% g l u t a r a l d e h y d e in M S B f o r i0 min, r i n s e d w e l l w i t h M S B a n d air dried. The membrane fragments were then s t a i n e d w i t h 2% a q u e o u s u r a n y l a c e t a t e a n d e x a m i n e d in a P h i l i p s 300 e l e c t r o n m i c r o : scope. P h o t o g r a p h i c p r i n t s of r a n d o m l y sel e c t e d r e g i o n s of m e m b r a n e w e r e a n a l y z e d u s i n g s t a n d a r d a r e a s to d e t e r m i n e the freq u e n c y of c o a t e d v e s i c l e s . F i g u r e 5 h a s b e e n i n c l u d e d in t h i s p a p e r to permit a visual comparison of p l a s m a m e m branes from protoplasts of s u s p e n s i o n cultured cells and leaf protoplasts. Details
r e g a r d i n g the p r e p a r a t i o n of t h e m e m b r a n e illustrated in F i g u r e 5 as w e l l as i n f o r m a tion regarding coated vesicle frequency have been previously reported (Van d e r V a l k et al. 1980, Van d e r V a l k a n d F o w k e 1981). T h e c e l l suspension used by Van der Valk and co-workers is g e n e t i c a l l y s i m i l a r to the l e a f m a t e r i a l u s e d in the c u r r e n t s t u d y s i n c e it w a s originally d e r i v e d f r o m t i s s u e of a l b i n o seedlings (Su/su). P r o t o p l a s t s from albino leaves could not be obtained for this study b e c a u s e of t e c h n i c a l d i f f i c u l t i e s . RESULTS
AND
DISCUSSION
Cultured leaf protoplasts exhibited a charact e r i s t i c lag p e r i o d b e f o r e r e g e n e r a t i n g suff i c i e n t c e l l w a l l to b e d e t e c t e d b y C a l c o fluor staining (see F o w k e a n d G a m b o r g 1980, W i l l i s o n a n d K l e i n 1982). A f t e r 4 hr c u l t u r e most protoplasts w e r e d e v o i d of d e t e c t a b l e
Fig. 1 Light micrograph showing Calcofluor p o s i t i v e f i b r i l s on t h e s u r f a c e of t o b a o c o l e a f protoplasts c u l t u r e d f o r 8 hr. b a r : 30 ~m. Fig. 2 Electron micrograph showing a negatively s t a i n e d f r a g m e n t of t o b a c c o l e a f p l a s m a m e m b r a n e . Coated vesicles (double arrows), fine filam e n t s ( s i n g l e a r r o w s ) and a m i c r o t u b u l e (Mt) are p r e s e n t , b a r : 200 nm. Fig. 3 Electron micrograph of a n e g a t i v e l y s t a i n e d f r a g m e n t of t o b a c c o l e a f p l a s m a m e m b r a n e s h o w i n g a p a t c h of a l v e o l a t e c o a t m a t e r i a l , b a r = 200 nm. Fig. 4 Electron micrograph of a f r a g m e n t of t o b a c c o leaf p l a s m a m e m b r a n e s h o w i n g d i s t r i b u t i o n of c o a t e d v e s i c l e s (arrows).bar = 0.5 ~m. Fig. 5 Electron micrograph of a f r a g m e n t of p l a s m a m e m b r a n e f r o m s u s p e n s i o n c u l t u r e d t o b a c c o cell showing distribution of c o a t e d v e s i c l e s (single arrows). N o t e the b u n d l e of m i c r o t u b u l e s ( d o u b l e a r r o w ) , b a r : 0.5 ~m ( k i n d l y s u p p l i e d by P. V a n d e r Valk).
294 Calcofluor s t a i n i n g b u t b y 6 hr p r o t o p l a s t s were enclosed within a faintly staining fibr i l l a r n e t w o r k . By 8 hr an e x t e n s i v e n e t w o r k of C a l c o f l u o r - p o s i t i v e fibrils was observed on the m a j o r i t y of p r o t o p l a s t s (Fig. i). A f t e r 5 d a y s in c u l t u r e the f i b r i l l a r n e t w o r k h a d b e e n r e p l a c e d by a u n i f o r m l y fluores c e n t c e l l wall. T h e n a t u r e of t h e f i b r i l s on the c u l t u r e d l e a f p r o t o p l a s t s has n o t as y e t been established. Calcofluor a l o n e is n o t a r e l i a b l e i n d i c a t o r for c e l l u l o s e b e c a u s e it can s t a i n o t h e r p o l y s a c c h a r i d e s (see W i l l i s o n and K l e i n 1982). F i b r i l s h a v e b e e n d e m o n s t r a t e d on the s u r f a c e of c u l t u r e d p r o t o p l a s t s b y a v a r i e t y of t e c h n i q u e s (e.g., n e g a t i v e staining, freeze-etching, replicas) and recent biochemical evidence suggests that they a r e c o m p o s e d of c e l l u l o s e (e.g., K l e i n et al. 1981). C o a t e d v e s i c l e s w e r e o b s e r v e d on the i n n e r s u r f a c e of the p l a s m a m e m b r a n e of b o t h S u / s u heterozygous and n o r m a l p l a n t s . T h e i r s i z e a n d s t r u c t u r e a p p e a r e d s i m i l a r to t h a t r e p o r t e d f o r c o a t e d v e s i c l e s on p l a s m a m e m branes from suspension cultured tobacco cells (Fig. 2 cf. V a n d e r V a l k a n d F o w k e 1981). It is i m p o r t a n t to r e a l i z e t h a t a n u m b e r of the structures identified in t h i s p a p e r as c o a t e d vesicles, are in f a c t c o a t e d p i t s ( v e s i c l e s incorporated i n t o the p l a s m a m e m b r a n e ) . However, it is n o t p o s s i b l e to d i s t i n g u i s h t h e s e two s t r u c t u r e s in s u r f a c e v i e w s of the plasma membrane. Occasionally large regions of a l v e o l a t e c o a t w e r e o b s e r v e d on t h e p l a s m a membrane (Fig. 3). S i m i l a r s t r u c t u r e s h a v e been reported previously for both plant and animal plasma membranes (Van d e r V a l k a n d F o w k e 1981, A g g e l e r a n d W e r b 1982, M a u p i n a n d P o l l a r d 1983) b u t t h e i r s i g n i f i c a n c e is unknown. R e s u l t s of t h r e e s e p a r a t e e x p e r i m e n t s revealed average frequencies ~f 1.54, 0 . 6 6 and 0 . 6 7 c o a t e d v e s i c l e s p e r Bm of p l a s m a m e m b r a n e ( T a b l e i). The n u m b e r of c o a t e d 2 v e s i c l e s p e r ~m r a n g e d f r o m 0 . 2 9 to 2.70 o v e r the t h r e e e x p e r i m e n t s . The d i f f e r e n c e in the a v e r a g e n u m b e r of c o a t e d v e s i c l e s o b served between experiment 1 and the o t h e r two e x p e r i m e n t s m a y b e d u e to v a r i a t i o n s in the age of l e a v e s used. T h e l e a v e s f o r experiment 1 were approximately 6-8 cm in l e n g t h w h i l e t h o s e f o r the o t h e r e x p e r i m e n t s e x c e e d 13 cm in l e n g t h . D e s p i t e the v a r i a b i -
lity observed, it is c l e a r t h a t the f r e q u e n c y of c o a t e d v e s i c l e s On l e a f p l a s m a m e m b r a n e s is c o n s i d e r a b l y l o w e r t h a n on p l a s m a m e m b r a n e s of p r o t o p l a s t s derived from suspension c u l t u r e d t o b a c c o c e l l s (SU 677). V a n d e r V a l k a n d F o w k e (1981) r e p o r t e d an a v e r a g e of 8.0 c o a t e d v e s i c l e s p e r ~m of m e m b r a n e w i t h a r a n g e of 3-12 p e r ~m of m e m b r a n e . Figure 4 and 5 illustrate the d i f f e r e n c e in c o a t e d v e s i c l e f r e q u e n c y b e t w e e n the two t y p e s of p r o t o plasts. The significance of t h i s d i f f e r e n c e is u n c l e a r at p r e s e n t . C o a t e d v e s i c l e s m a y h a v e "a r o l e in p l a n t c e l l w a l l d e p o s i t i o n (see N e w c o m b 1980) a n d t h e y are o f t e n l o c a l i z e d in r e g i o n s of a c t i v e c e l l w a l l f o r m a t i o n s u c h as f o r m i n g c e l l p l a t e s ( F r a n k e a n d H e r t h 1974, N a k a m u r a a n d M i k i - H i r o s h i g e 1982) a n d the t i p s of a c t i v e l y g r o w i n g r o o t h a i r s ( R o b e r t s o n a n d L y t t l e t o n 1982) a n d c o t t o n h a i r s ( R y s e r 1979). If c o a t e d v e s i c l e s h a v e a r o l e in t r a n s p o r t i n g m a t e r i a l s to the r e generating cell wall, cell culture protop l a s t s w h i c h a r e c a p a b l e of r a p i d i n i t i a t i o n of n e w w a l l w o u l d be e x p e c t e d to h a v e m o r e coated vesiclesassociated with their plasma membrane than leaf protoplasts which experience a pronounced lag p r i o r to w a l l f o r m a tion. C e l l w a l l f o r m a t i o n in P l a n t s i n v o l v e s t h e t r a n s p o r t of m a t r i x m a t e r i a l s to the c e l l surface via Golgi vesicles which fuse with and contribute to the p l a s m a m e m b r a n e . It is conceivable that coated vesicles might funct i o n by r e c y c l i n g p l a s m a m e m b r a n e b a c k i n t o the c y t o p l a s m . A h i g h e r f r e q u e n c y of c o a t e d v e s i c l e s on p l a s m a m e m b r a n e s of s u s p e n s i o n culture protoplasts would be consistent with t h i s i d e a as w e l l . Recent evidence suggests that coated vesicles m i g h t h a v e a r o l e in the p r o c e s s of e n d o c y t o s i s by p l a n t s ( T a n c h a k et al. 1 9 8 3 ) . T h e difference in c o a t e d v e s i c l e f r e q u e n c i e s obs e r v e d in the p r e s e n t s t u d y m i g h t t h e r e f o r e reflect general differences in m e t a b o l i c a c t i v i t i e s of p r o t o p l a s t s from rapidly growing suspension cultured cells and mature leaf cells. very few microtubules w e r e o b s e r v e d on l e a f plasma membranes (Fig. 2, 4). In c o n t r a s t , plasma membrane fragments from protoplasts c a p a b l e of a c t i v e w a l l s e c r e t i o n a r e c h a r a c terized by numerous microtubules (Van d e r V a l k et al. 1980). T h e d i r e c t c o r r e l a t i o n
T a b l e i. Frequency Leaf Protoplasts
Membrane
of
Coated
Vesicles
Number Experiment 1
2
3
aRange
Fragments
from
Nicotiana
Examined
T o t a l A r e a o~ Membrane (~m -)
A v e r a g e N u m b e r of Coated Vesicles per
Heterozygous p l a n t s (Su/su)
ii
i158
1.54
(0.44
- 2.70) a
Heterozygous p l a n t s (Su/su)
9
808
0.66
(0.35
- 1.21)
15
1060
0.67
(0.29
- 1.14)
Normal plants
given
Plasma
(su/su)
in p a r e n t h e s e s .
tabacum
of
Protoplasts
Leaf Source
on
~m
2
295 b e t w e e n p r e s e n c e of m i c r o t u b u l e s on the p l a s m a m e m b r a n e and a b i l i t y to s y n t h e s i z e c e l l w a l l is p e r h a p s not s u r p r i s i n g s i n c e m i c r o t u b u l e s s e e m to h a v e an i m p o r t a n t r o l e in o r i e n t i n g c e l l u l o s e m i c r o f i b r i l d e p o s i t i o n ( r e v i e w e d by G u n n i n g and H a r d h a m 1982 Fowke et al. 1983). F i n e f i l a m e n t s w e r e o f t e n o b s e r v e d on f r a g m e n t s of leaf p l a s m a m e m b r a n e (Fig. 2 The n a t u r e of t h e s e f i l a m e n t s is p r e s e n t l y u n k n o w n . It is p o s s i b l e that t h e y are c o m p o s e d of a c t i n s i n c e a c t i n m i c r o f i l a m e n t s h a v e b e e n i d e n t i f i e d in b o t h a l g a l and h i g h e r p l a n t p r o t o p l a s t s ( M a r c h a n t 1976, Y a m a g u c h i and N a g a i 1981). A l t e r n a t i v e l y , t h e y m a y not be c o m p o s e d of a c t i n and m a y r e p r e s e n t p a r t of a f i l a m e n t o u s n e t w o r k b e l i e v e d to p r o v i d e s u p p o r t f o r the n u c l e u s in p l a n t p r o t o p l a s t s ( P o w e l l et al. 1982).
ACKNOWLEDGEMENTS
The a s s i s t a n c e of Mr. J. S h y l u k is g r e a t l y appreciated. This project was supported by the N a t u r a l S c i e n c e s a n d E n g i n e e r i n g R e s e a r c h C o u n c i l of C a n a d a .
REFERENCES
A g g e l e r J, W e r b Z (1982) J C e l l B i o l 95: 613-623 D o o h a n ME, P a l e v i t z BA (1980) P l a n t a 149: 389-401 F o w k e LC (1983) In: G a r r o d D, C h a d w i c k C (eds) R e c e p t o r s in p l a n t s and s l i m e m o u l d s , M a r c e l D e k k e r , New York, (in press) F o w k e LC, C o n s t a b l e F (1982) In: G i l e s KL, Sen SK (eds) P l a n t c e l l c u l t u r e in c r o p improvement, Plenum Publishing Corporation, N e w York, pp 1 8 5 - 2 0 0 F o w k e LC, G a m b o r g OL (1980) Int Rev C y t o l 68: 9-51
F o w k e LC, G r i l l i n g LR, M e r s e y BG, Van d e r V a l k P (1983) P r o c e e d 6th Int P r o t o p l a s t Symp, B a s e l S w i t z e r l a n d F r a n k e WW, H e r t h W (1974) E x p t l C e l l Res 89: 447-451 G u n n i n g BES, H a r d h a m AR (1982) Ann Rev P l a n t P h y s i o l 33: 6 5 1 - 6 9 8 Kao KN (1982) In: W e t t e r LR, C o n s t a b e l F (eds) P l a n t t i s s u e c u l t u r e m e t h o d s , N a t i o nal R e s e a r c h C o u n c i l of C a n a d a , S a s k a t o o n , pp 49-56 K l e i n AS, M o n t e z i n o s D, D e l m e r DP (1981) P l a n t a 152: 1 0 5 - 1 1 4 L e o n a r d RT, H o d g e s T K (1980) In: T o l b e r t NE (ed) T h e b i o c h e m i s t r y of p l a n t s , A c a d e m i c Press, New York, pp 1 6 3 - 1 8 2 L l o y d CW, S l a b a s AR, P o w e l l AJ, L o w e SB (1980) P l a n t a 147: 5 0 0 - 5 0 6 M a r c h a n t HJ (1976) P l a n t a l 3 1 : 1 1 9 - 1 2 0 M a r c h a n t HJ (1978) E x p t l C e l l Res 115: 2 5 - 3 0 M a u p i n P, P o l l a r d TD (1983) J C e l l B i o l 96: 51-62 M u r a s h i g e T, S k o o g F (1962) P h y s i o l P l a n t 15: 431-497 N a k a m u r a S, M i k i - H i r o s h i g e H (1982) J U l t r a s t r u c Res 80: 3 0 2 - 3 1 1 N e w c o m b EH (1982) In: O c k l e f o r d CJ, W h y t e A (eds) C o a t e d v e s i c l e s , C a m b r i d g e U n i v e r s i t y Press, New York, pp 5 5 - 6 8 P o w e l l AJ, P e a c e GW, S l a b a s AR, L l o y d C W (1982) J C e l l Sci 56: 3 1 9 - 3 3 5 R o b e r t s o n JG, L y t t l e t o n P (1982) J C e l l Sci 58: 6 3 - 7 8 R y s e r U (1979) P r o t o p l a s m a 98: 2 2 3 - 2 3 9 T a n c h a k M, G r i f f i n g LR, M e r s e y BG, F o w k e LC (1983) J C e l l B i o l ( A b s t r a c t ) Van d e r V a l k P, F o w k e LC (1981) C a n J Bot 59: 1307-1313 Van d e r V a l k P, R e n n i e PJ, C o n n o l l y JA, F o w k e LC (1980) P r o t o p l a s m a 105: 2 7 - 4 3 W i l l i s o n JHM, K l e i n AS (1982) In: B r o w n R M (ed) C e l l u l o s e and o t h e r n a t u r a l p o l y m e r s y s t e m s , P l e n u m , N e w Y o r k , pp 6 1 - 8 5 Y a m a g u c h i Y, N a g a i R (1981) J C e l l Sci 48: 193-205
