Biogenesis of Cellulose Microfibrils and the Role of Microtubules in

Chapter 19

Biogenesis of Cellulose Microfibrils and the Role of Microtubules in Green Algae Takao Itoh

Downloaded by MONASH UNIV on December 5, 2014 | http://pubs.acs.org Publication Date: July 31, 1989 | doi: 10.1021/bk-1989-0399.ch019

Wood Research Institute, Kyoto University, Uji, Kyoto 611, Japan

This chapter reviews our knowledge of factors controlling cellulose deposition in green algae. Firstly, the types of cellulose synthesizing particle complexes in green algae are discussed. Secondly, new evidence on the oriention of microtubules in selected giant marine algae and their relationship to the orientation of cellulose microfibrils is presented. Based on this information, a mechanism for the assembly of cellulose microfibrils in giant marine algae is proposed. O u r c u r r e n t u n d e r s t a n d i n g o f cellulose m i c r o f i b r i l biogenesis a n d assemb l y comes f r o m (a) freeze-fracture studies o f the p l a s m a m e m b r a n e o f cells a c t i v e l y p r o d u c i n g cellulose m i c r o f i b r i l s ; (b) observations o f m i c r o t u b u l e s by immunofluorescence m i c r o s c o p y ; (c) direct i m a g i n g o f cellulose m i c r o f i b rils (1-5); a n d (d) in vitro synthesis o f cellulose u s i n g b a c t e r i a l cell m e m brane p r e p a r a t i o n s (6). T h i s chapter examines recent progress i n freezefracture a n d immunofluorescence studies o n the biogenesis o f cellulose m i crofibrils, as w e l l as addressing the role o f m i c r o t u b u l e s i n several green algae. F o r t h e last decade, m u c h o f our knowledge o f the s t r u c t u r e a n d f u n c t i o n o f cellulose f o r m i n g enzyme complexes (so-called T e r m i n a l C o m p l e x e s or T C ' s ) has been based o n results o b t a i n e d f r o m freeze-fracture studies. T h e m a i n discoveries f r o m these studies were (a) the existence o f l i n e a r T C ' s by B r o w n a n d M o n t e z i n o s (7) i n Oocystis apiculata, a n d (b) t h e occurrence o f rosette T C ' s by G i d d i n g s et ai (8) i n Micrasterias denticulata. A s w i l l be discussed later, the occurrence o f these t w o types o f cellulose s y n t h e s i z i n g complexes has some e v o l u t i o n a r y significance as regards c u r rent phylogenetic r e l a t i o n s h i p s w i t h i n the p l a n t k i n g d o m . T h i s is because a l l l a n d p l a n t s , i n c l u d i n g higher p l a n t s , mosses a n d ferns, have rosettes (911), whereas some algae have linear T C ' s a n d others d o n o t . It has been 0097-6156/89/0399-0257$06.00A) © 1989 American Chemical Society

In Plant Cell Wall Polymers; Lewis, N., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1989.


258

PLANT C E L L W A L L P O L Y M E R S

proposed t h a t the T C ' s move o n the fluid p l a s m a m e m b r a n e b y forces generated d u r i n g c r y s t a l l i z a t i o n of cellulose m i c r o f i b r i l s (12,13). In green algae, the n e w l y synthesized m i c r o f i b r i l s first have a r a n d o m o r i e n t a t i o n d u r i n g the synthesis of the p r i m a r y w a l l a n d then a n ordered o r i e n t a t i o n d u r i n g secondary w a l l f o r m a t i o n . It is t h u s conceivable t h a t the factors c o n t r o l l i n g the o r i e n t a t i o n of T C ' s i n green algae m a y also be responsible for the o r i e n t a t i o n of m i c r o f i b r i l s ; t h i s c o u l d be achieved, for e x a m p l e , by c h a n n e l l i n g c y t o p l a s m i c m i c r o t u b u l e s . T h i s view is presented because i n higher p l a n t s , a n d some algal cells, rosette particles are a p p a r e n t l y channelled b y c y t o p l a s m i c m i c r o t u b u l e s w h i c h r u n p a r a l l e l to one another (14-16). However, as a counter to t h i s a r g u m e n t , some green algae have l i n e a r T C ' s w h i c h do not appear to have m i c r o f i b r i l l a r o r i e n t a t i o n c o n t r o l l e d b y m i c r o t u b u l e s d u r i n g secondary w a l l synthesis (17). T h i s evidence suggests therefore t h a t there m a y be different mechanisms c o n t r o l l i n g m i c r o f i b r i l l a r o r i e n t a t i o n between higher a n d lower plants. Structure of T C ' s in G r e e n Algae T h e green algae i n c l u d e b o t h C h l o r o p h y t a a n d C h a r o p h y t a . T h e T C ' s of C h l o r o p h y t a have been observed i n four orders of C h l o r e l l a l e s , C l a d o p h o rales, S i p h o n o c l a d a l e s , a n d Z y g n e m a t a l e s , a n d those of C h a r o p h y t a i n one order of C h a r ales (Table I). Recent investigations show t h a t 17 genera a n d 23 species out of three orders of C h l o r e l l a l e s , C l a d o p h o r a l e s a n d S i p h o n o cladales a l l have l i n e a r T C ' s , as shown i n T a b l e I. W h i l e linear T C ' s have been observed i n some freshwater algae such as Oocystis (7,18), Eremosphaera (19), a n d Glaucocystis (20) species, most are f o u n d i n m a r i n e - t y p e algae such as Boergesenia (21,22), Boodlea ( F i g . 1) (23,24), Dictyosphaeria ( F i g . 2), Ernodesmis ( F i g . 3), Microdictyon (19), Siphonocladus ( F i g . 4), Struvea ( F i g . 5), Valonia ( F i g . 6) (17,24,25), Valoniopsis ( F i g . 7), a n d Chaetomorpha ( F i g . 8) (19,25) species; these eight genera belong t o the Siphonocladales. A l t h o u g h Chlorellales, Cladophorales and Siphonocladeles have linear T C ' s , there are significant differences i n their l o c a t i o n s ; t h a t is, C h l o r e l l a l e s have T C ' s o n l y on the E - f r a c t u r e face, w h i l e b o t h C l a d o p h o r a l e s a n d Siphonocladales have T C ' s on b o t h E - a n d P - f r a c t u r e faces, thereby m a k i n g t r a n s m e m b r a n e particles. A d d i t i o n a l l y , the T C s t r u c t u r e of Z y g n e m a t a l e s is quite different f r o m those of the other three orders i n C h l o r o p h y t a . F o u r genera of the Z y g n e m a t a l e s , i n c l u d i n g Closterium (16,26), Micrasterias (8,27,28), Mougeotia (29), a n d Spirogyra (19,30) species have been investigated so far. A l l have rosettes o n l y o n the P - f r a c t u r e face. T h e cells can have either r a n d o m a n d / o r u n i d i r e c t i o n a l rosette d i s t r i b u t i o n s d u r i n g active synthesis of the p r i m a r y w a l l , a n d hexagonal arrays d u r i n g the synthesis of the secondary wall. M o r e recently, rosettes have been observed i n Chara sp. (31) a n d Nitella translucens (32), w h i c h belong to another s u b d i v i s i o n , C h a r o p h y t a . T h e rosettes i n t h i s species occur separately w i t h o u t m a k i n g any p o l y g o n a l arrays as f o u n d i n Z y g n e m a t a l e s . T h i s suggests t h a t C h a r a l e s are closer to vascular p l a n t s t h a n the other algae, i f plant phylogenic classifications



19.

ITOH

Biogenesis of Cellulose Microfibrils

Figure 1. TCs of Boodlea composita on P-fracture face.

Figure 2. TCs of Dictyosphaeria cavernosa on P-fracture face.

Figure 3. TCs of Ernodesmis verticillata on Ε-fracture face.


259


260

PLANT C E L L W A L L

POLYMERS

Figure 4. TC's of Siphonocladus tropicus on P-fracture face.

Figure 5. TC's of Struvea elegans on P-fracture face.

Figure 6. TC's of Valonia ventricosa on Ε-fracture face.



19.

ITOH


Figure 7. T C s of Valoniopsis pachynema on P-fracture face.

Figure 8. T C s of Chaetomorpha

auricoma on P-fracture face.


261

262

PLANT C E L L W A L L P O L Y M E R S

T a b l e I. C u r r e n t S u m m a r y of the S t r u c t u r e a n d L o c a t i o n of T C s a m o n g Green Algae


S u b d i v i s i o n , O r d e r , Species Chlorophyta Chlorellales Eremosphaera sp. Glaucocysiis nostochinearum Oocystis apiculata Oocystis solitaria Cladophorales Chaetomorpha sp. Chaetomorpha aerea Chaetomorpha moniligera Siphonocladales Boergesenia forbesii Boodlea coacta Boodlea composita Dictyosphaeria cavernosa Ernodesmis verticillata Siphonocladus tropicus Struvea elegans Valonia macrophysa Valonia ventricosa Valonia ventricosa Zygnematales Closterium acerosum Closterium sp. Micrasterias cruxmelitensis Micrasterias denticulata Mougeotia sp. Spirogyra sp. Charophyta Charales Chara sp. Nitella translucens

TC Structure

TC Location

Linear Linear Linear Linear

EF EF EF EF

TC TC TC TC

only only only only

Linear T C Linear T C Linear T C

EF k PF EF k PF EF k PF

Linear Linear Linear Linear Linear Linear Linear Linear Linear Linear

EF EF EF EF EF EF EF EF EF EF

TC TC TC TC TC TC TC TC TC TC

k k k k k k k k k k

PF PF PF PF PF PF PF PF PF PF

Rosettes Rosettes Rosettes Rosettes Rosettes Rosettes

PF PF PF PF PF PF

only only only only only only

Rosettes Rosettes

P F only P F only

Reference

19 20 7 18 19 24 25 21,22 23 24 T h i s chapter T h i s chapter T h i s chapter T h i s chapter 17 24,25 T h i s chapter 26 16 28 27,8 29 30,19

31 32

can be m a d e based u p o n cellulose s y n t h e s i z i n g complexes. It c o u l d t h u s be argued f r o m these d a t a t h a t the e v o l u t i o n of T C ' s w i l l follow the lines envisaged i n F i g . 9, w h i c h is a m o d i f i c a t i o n of a scheme proposed b y H e r t h (11) for the h y p o t h e t i c a l e v o l u t i o n a r y lines of p u t a t i v e cellulose s y n t h e s i z i n g complexes. B a s i c a l l y , t h i s stems f r o m the fact t h a t b o t h rosettes a n d l i n e a r T C ' s are composed of c o m m o n p a r t i c l e s u b u n i t s s i m i l a r i n size; rosettes consist of s i x particles, each h a v i n g an 8 n m d i a m e t e r of (8) a n d l i n e a r T C ' s consists of three rows w i t h an average d i m e n s i o n of ca. 8 n m for each i n d i v i d u a l p a r t i c l e (25). However, T C ' s have o n l y been f o u n d i n a l i m i t e d



19.

ITOH


263

n u m b e r of species a m o n g the algae. Consequently, a further survey of T C ' s is necessary to develop a detailed phylogenic hypothesis. T C ' s n o r m a l l y occur at the end of m i c r o f i b r i l i m p r i n t s o n either E - or P - f r a c t u r e faces of the p l a s m a m e m b r a n e . E a c h i m p r i n t does not necessarily correspond to a single m i c r o f i b r i l , but often to bundles of t h e m . D u r i n g the d e p o s i t i o n of r a n d o m m i c r o f i b r i l s of the p r i m a r y w a l l , the i m p r i n t s r u n a l o n g the p l a s m a m e m b r a n e , a n d often w i t h c u r v e d t r a i l s . O n the other h a n d , w h e n ordered m i c r o f i b r i l s of the secondary w a l l are s y n t h e s i z e d , the i m p r i n t s r u n straight a n d p a r a l l e l to one another. In Oocystis species, each p a i r e d T C runs i n an opposite d i r e c t i o n (31). In most giant m a r i n e algae, some T C ' s appear to r u n i n one d i r e c t i o n , w h i l e others r u n i n the opposite d i r e c t i o n d u r i n g the synthesis of ordered m i c r o f i b r i l s . T h e l a t t e r case is i l l u s t r a t e d i n F i g u r e 10 w h i c h shows the Ε-fracture face of n e w l y f o r m e d cellulose m i c r o f i b r i l s of ordered o r i e n t a t i o n . T h e T C n u m b e r e d " 1 " is m o v i n g to the right whereas the T C n u m b e r e d " 2 " is m o v i n g to the left. C l e a r l y , i f the i n d i v i d u a l T C synthesizes u n i d i r e c t i o n a l g l u c a n chains of / ? - l , 4 linkages, the cell w a l l s h o u l d have a n a n t i - p a r a l l e l g l u c a n c h a i n orientation. Development of Linear T C ' s i n Selected G r e e n Algae T h e l e n g t h of linear T C ' s is variable a n d depends o n the d e v e l o p m e n t a l phase of cell g r o w t h . T h i s was determined f o l l o w i n g w o u n d i n g of the m o t h e r cells of B. forbesii, where the aplanospores were regenerated w i t h i n 1.5 h , a n d the p r i m a r y a n d the secondary walls were synthesized 2 to 4h a n d 4 to 5h after w o u n d i n g , respectively (33). F i g u r e 11 shows the effect of t i m e o n T C l e n g t h f o l l o w i n g aplanospore i n d u c t i o n i n Boergesenia forbesii. A s can be seen, the T C length increases o n l y d u r i n g f o r m a t i o n of the p r i m a r y w a l l ; no further increase occurs after deposition of the secondary w a l l . However, w h e n we look closer at the freeze-fracture r e p l i c a of the aplanospores i n B. forbesii j u s t before, or at the t i m e of, synthesis of cellulose m i c r o f i b r i l s , m a n y nascent T C ' s can be observed on the P - f r a c t u r e face of the p l a s m a m e m b r a n e ( F i g . 12). T h e smallest T C observed h a d o n l y 10 p a r t i c l e s . M o s t nascent T C ' s d i d not show a d i r e c t i o n a l arrangement of p a r t i c l e s , w h i l e some h a d already organized an elongated cluster (double arrows i n F i g . 12). T h e m e a n length of the T C ' s i n t h i s phase was 114 n m . T h e nascent T C ' s increased i n length d u r i n g the synthesis of the p r i m a r y w a l l , u n t i l the ordered m i c r o f i b r i l s were assembled. T h e l e n g t h of nascent T C ' s contrasted w i t h t h a t of f u l l y elongated T C ' s , w h i c h h a d a m a x i m u m l e n g t h of ca. 1 m (mean l e n g t h : 665 n m ) i n 20h cells of B. forbesii ( F i g . 13). Orientation of C o r t i c a l Microtubules T h e cellulose s y n t h e s i z i n g enzyme complexes i n green algae can be d i v i d e d i n t o rosettes a n d linear T C ' s ; the latter increases i n size d u r i n g c i r c u m f e r e n t i a l e x p a n s i o n of the cells. T h e movement of b o t h g r o w i n g a n d m a t u r e T C ' s i n the p l a s m a m e m b r a n e is controlled by forces generated b y c r y s t a l l i z a t i o n of m i c r o f i b r i l s , leaving the h i g h l y c r y s t a l l i n e m i c r o f i b r i l s i n their wake (7,34).


264

PLANT C E L L W A L L POLYMERS

HIGHER

PLANTS

MOSSES & FERNS

CHARALES

SIPHONOCLADALES CLADOPHORALES · · · · · · · · ·

ZYGNEMATALES

CHLORELALES


\

ALGAE

\

(ROSETTES)

(LINEAR

\/

....... FUCALES

TCS)

F i g u r e 9. H y p o t h e t i c a l i l l u s t r a t i o n for e v o l u t i o n a r y t r e n d o f p u t a t i v e cel lulose s y n t h e s i z i n g e n z y m e complexes f r o m algae t o higher p l a n t s . B o t h rosettes a n d linear T C ' s originate f r o m their c o m m o n s u b u n i t of 8 n m p a r ticle. A s t e r i s k (*) represents a rosette.

F i g u r e 10. Ε-fracture face o f the p l a s m a m e m b r a n e d u r i n g active synthesis of ordered m i c r o f i b r i l s i n secondary w a l l o f Valonia macrophysa. Imprints of m i c r o f i b r i l s r u n p a r a l l e l t o one another. T C ' s numbered " 1 " a n d " 2 " direct opposite ways t o one another.



19.

ITOH


F i g u r e 11. T i m e course of T C length i n the aplanospores of forbesii.


265

Boergesenia


266


F i g u r e 12. F o u r nascent T C ' s (arrowheads) are s h o w n on P - f r a c t u r e face of the p l a s m a m e m b r a n e i n 2h aplanospore of Boergesenia forbesii after wounding.

F i g u r e 13. T w o f u l l y elongated T C ' s (ca. 1 m ) are s h o w n o n P - f r a c t u r e face of the p l a s m a m e m b r a n e i n 20h cell of Boergesenia forbesii.



19.

ITOH


267

Since the m a t u r e walls of Valonia a n d Boergesenia have a crossed lamellate s t r u c t u r e (17,35-37), the d i r e c t i o n of T C movement m u s t be c o n t r o l l e d i n some u n k n o w n m a n n e r . O n the other h a n d , giant m a r i n e a l gae, w h i c h also have linear T C ' s , but of a p p a r e n t l y r a n d o m o r i e n t a t i o n , o n l y synthesize m i c r o f i b r i l s of r a n d o m o r i e n t a t i o n on the i n n e r m o s t face of the p l a s m a m e m b r a n e d u r i n g the synthesis of the p r i m a r y w a l l . It was, therefore, t i m e l y to e x a m i n e whether c o r t i c a l m i c r o t u b u l e s were r e s p o n sible for o r i e n t a t i o n of m i c r o f i b r i l s , since recent investigations suggested t h a t the d i r e c t i o n of m i c r o f i b r i l d e p o s i t i o n i n higher p l a n t cells (38-40) a n d some algae (Oocystis a n d Micrasterias (8,16,33)), was c o n t r o l l e d b y c o r t i c a l m i c r o t u b u l e s . However, i n the a l g a Closterium, microtubules functioned o n l y to l i m i t cellulose synthesis to a localized region (41). A d d i t i o n a l l y , freeze-fracture studies suggested t h a t newly synthesized m i c r o f i b r i l s i n the spherical cells of the giant m a r i n e algae Valonia macrophysa were not p a r allel to the u n d e r l y i n g m i c r o t u b u l e s (17). In the light of these c o n t r a d i c t o r y findings, the role of m i c r o t u b u l a r o r i e n t a t i o n i n cellulose m i c r o f i b r i l l a r o r i e n t a t i o n a m o n g green algae was r e - e x a m i n e d u s i n g immunofluorescence microscopy. L l o y d et al. (43) first used this technique to observe c o r t i c a l m i c r o t u b u l e s , following the pioneering work o n p l a n t cells b y F r a n k e (42). Since t h e n , it has been used m a n y times to show m i c r o t u b u l e o r i e n t a t i o n over whole cells (44). T h e m a t e r i a l s used for the immunofluorescent s t a i n i n g were a p l a n o spores of B. forbesii a n d V. ventricosa, where cellulose m i c r o f i b r i l o r i e n t a t i o n c o u l d easily be s h o w n by s t a i n i n g w i t h the fluorescent b r i g h t e n i n g agents C a l c o f l u o r a n d T i n o p a l L P W (34). A s m e n t i o n e d i n the previous section, Boergesenia aplanospores synthesized r a n d o m m i c r o f i b r i l s between 2 a n d 4 h after w o u n d i n g of m o t h e r cells a n d ordered m i c r o f i b r i l s after 4 h . T h e Boergesenia aplanospores i n w h i c h s p h e r a t i o n h a d j u s t been c o m p l e t e d showed r a n d o m l y oriented m i c r o t u b u l e s j u s t under the p l a s m a m e m b r a n e ( F i g . 14), a n d the aplanospores i n 3h p o s t - w o u n d i n g d i d not differ f r o m those at 1.5h. ( F i g . 15). However, after 6h p o s t - w o u n d i n g , d u r i n g w h i c h t i m e the n o r m a l l y synthesized ordered m i c r o f i b r i l s were deposited, o n l y r a n d o m l y oriented m i c r o t u b u l e s were observed under the thickened cell w a l l ( F i g . 16). A t a later phase of cell w a l l regeneration, immunofluorescent s t a i n i n g of m i c r o t u b u l e s b y a n t i g e n - a n t i b o d y reactions became m o r e difficult because of the thickened wall s u r r o u n d i n g the aplanospores. N e v e r theless, we m a n a g e d to observe the arrangement of m i c r o t u b u l e s even after 8, 10, a n d 20h p o s t - w o u n d i n g w i t h o u t h a v i n g to resort to e n z y m a t i c cell w a l l digestion. In a l l cases, o n l y r a n d o m m i c r o t u b u l a r o r i e n t a t i o n p a t t e r n s ( F i g s . 17, 18 a n d 19) were observed. However, after successive c u l t u r e of the spherical aplanospores for m o r e t h a n five days, g e r m i n a t i o n occurred w i t h a t y p i c a l t i p g r o w t h to make r h i zoids (45); the o r i e n t a t i o n of m i c r o f i b r i l s i n a single e l o n g a t i n g r h i z o i d of 10 days p o s t - w o u n d i n g , for e x a m p l e , has been described recently (13). I n our s t u d y , the innermost w a l l lamellae i n the r h i z o i d showed three different orientations of m i c r o f i b r i l s , i.e., transverse, o b l i q u e , a n d l o n g i t u d i n a l to the g r o w i n g cell a x i s . F o l l o w i n g immunofluorescent s t a i n i n g of the m i c r o t u b u l e s



268


POLYMERS

Figure 14. Immunofluorescence micrograph of microtubule orientation during cell regeneration in Boergesenia forbesii 1.5 h after wounding.

Figure 15. Immunofluorescence micrograph of microtubule orientation during cell regeneration in Boergesenia forbesii 3 h after wounding.



19.

ITOH


269





270

PLANT C E L L WALL

POLYMERS





19.

ITOH


271

by the fluorescein isothiocyanate-conjugated a n t i b o d y , a n e l o n g a t i n g r h i z o i d of 8 days p o s t - w o u n d i n g showed ordered m i c r o t u b u l e s oriented l o n g i t u d i n a l l y to the g r o w i n g cell a x i s , as w e l l as p a r a l l e l to one another ( F i g . 20). It is n o t e w o r t h y t h a t l o n g i t u d i n a l m i c r o t u b u l e s were also observed i n the r o o t hairs of some higher p l a n t s w h i c h show t i p g r o w t h (46-48). In the case of V. ventricosa, s p h e r a t i o n was c o m p l e t e d w i t h i n 2h of w o u n d i n g , f o l l o w i n g w h i c h r a n d o m m i c r o f i b r i l s were regenerated between 5 a n d 12h a n d ordered m i c r o f i b r i l s w i t h i n 12 to 15h. T h e t i m e course of m i c r o t u b u l e o r i e n t a t i o n i n the aplanospores of V. ventricosa followed a t r e n d s i m i l a r to t h a t observed for B. forbesii; t h a t is, m i c r o t u b u l e s were a l w a y s oriented r a n d o m l y d u r i n g the synthesis of b o t h r a n d o m a n d ordered m i crofibrils. It is w o r t h n o t i n g the s t r u c t u r a l changes of m i c r o t u b u l e s i n the early phase of cell regeneration; the aplanospores p r o d u c e d f r o m V. ventricosa w i t h i n 2 a n d 3h of p o s t - w o u n d i n g d i d not show c o r t i c a l m i c r o t u b u l e s b u t i n s t e a d perinuclear m i c r o t u b u l e arrays ( F i g . 21), a n d the aplanospores after 4 h showed the i n i t i a t i o n of w a l l m i c r o t u b u l e s ( F i g . 22). F i g u r e s 2 3 a and 23b show 3h aplanospores of a c t i v e l y s y n t h e s i z i n g p r i m a r y w a l l p o i n t s . B o t h figures were t a k e n f r o m the same cell w i t h different focusing. F i g ure 23a was focused o n the nucleus; note t h a t several m i c r o t u b u l e s r a d i a t e f r o m the nucleus. F i g u r e 23b was focused o n the c o r t i c a l m i c r o t u b u l e s , where some of the perinuclear m i c r o t u b u l e s appeared as p a r t of the c o r t i cal m i c r o t u b u l e s . T h i s evidence suggests t h a t the nuclei m a y play a role as m i c r o t u b u l e o r g a n i z i n g centers ( M T O C ) d u r i n g the regeneration of the cell w a l l i n these green algae. Summary: entation

Microtubule-Independent

C o n t r o l of M i c r o f i b r i l O r i -

F r o m our immunofluorescence e x p e r i m e n t s , we have concluded t h a t (1) o n l y a r a n d o m m i c r o t u b u l a r o r i e n t a t i o n occurs d u r i n g regeneration of p r i m a r y a n d secondary walls a n d (2) the g r o w i n g r h i z o i d s d u r i n g t i p g r o w t h o n l y showed m i c r o t u b u l e s oriented l o n g i t u d i n a l l y to the g r o w i n g cell w a l l axis. However, since three different o r i e n t a t i o n s of m i c r o f i b r i l s i n the i n n e r most lamellae of the cell wall were observed, these findings suggest t h a t the d i r e c t i o n of movement of linear T C ' s was not controlled by c o r t i c a l m i c r o tubules, at least not i n the case of the two giant m a r i n e algae, Boergesenia and Valonia, s t u d i e d . L a C l a i r e (49) recently described a h i g h l y ordered a r r a y of p a r a l l e l a n d l o n g i t u d i n a l m i c r o t u b u l e s i n the coenocytic green algae Ernodesmis verticillata. However, w i t h other filamentous green algae, Boodlea coacta (50) and Chaetomorpha moniligera (51), aligned m i c r o t u b u l e s a n d m i c r o f i b r i l s were not always observed. It was thus suggested t h a t m i c r o f i b r i l o r i e n t a t i o n i n Siphonocladales and p r o b a b l y C l a d o p h o r a l e s m a y be independent of c y t o p l a s m i c m i c r o t u b u l e o r i e n t a t i o n . O n c e T C ' s are i n i t i a t e d i n the p l a s m a m e m b r a n e , they d i s t r i b u t e r a n d o m l y w i t h each T C r u n n i n g s t r a i g h t ahead. M o r e recent investigations showed t h a t the density of T C ' s was more or less constant (52), i n d i c a t i n g t h a t the n u m b e r of T C ' s d i d not increase a p p r e c i a b l y d u r i n g p r i m a r y w a l l



272


F i g u r e 20. Immunofluorescence m i c r o g r a p h of m i c r o t u b u l e o r i e n t a t i o n i n 8 d a y o l d cells of Boergesenia forbesii. H i g h l y ordered m i c r o t u b u l e s are oriented l o n g i t u d i n a l l y to the cell axis (double-headed a r r o w ) .

F i g u r e 21. Immunofluorescence m i c r o g r a p h of perinuclear m i c r o t u b u l e s i n the aplanospore of 3h p o s t - w o u n d i n g of Valonia venlricosa.



19.

ITOH


273

F i g u r e 22. Immunofluorescence m i c r o g r a p h of c o r t i c a l m i c r o t u b u l e s i n the aplanospore of 4h p o s t - w o u n d i n g of Valonia ventricosa.

F i g u r e 23. Immunofluorescence m i c r o g r a p h s i n the aplanospore of 3h postw o u n d i n g of Boergesenia forbesii. F i g u r e 23a is focused on the p e r i n u c l e a r m i c r o t u b u l e s , w h i l e F i g u r e 23b is focused on the c o r t i c a l m i c r o t u b u l e s w h i c h are o r i e n t e d r a n d o m l y .



274


POLYMERS

F i g u r e 24. O r i e n t a t i o n of m i c r o f i b r i l s is s h o w n i n 4h aplanospore of Boergesenia forbesii, stained w i t h fluorescent b r i g h t e n i n g agent T i n o p a l L P W .

F i g u r e 25. Freeze fractured replica. P-fracture face of the p l a s m a m e m b r a n e i n 15h aplanospore of Valonia ventricosa. T h e clustered T C ' s are s h o w n , suggesting the b u i l d - u p of new axis for m i c r o f i b r i l o r i e n t a t i o n .



19.

ITOH


275

synthesis, even t h o u g h a h i g h density o f T C ' s was often l o c a l i z e d i n some areas o f the p l a s m a m e m b r a n e . F i g u r e 24 shows the o r i e n t a t i o n o f m i c r o f i b rils i n aplanospores o f B. forbesii 4 h after w o u n d i n g , a n d s t a i n e d w i t h t h e fluorescent b r i g h t e n i n g agent T i n o p a l L P W . I n some areas, short s t r i a t i o n s of fluorescence merged into a center, suggesting a shift o f m i c r o f i b r i l o r i e n t a t i o n , p r o b a b l y b y localized membrane flow. T C l o c a l i z a t i o n c a n often be observed i n t h e t r a n s i t i o n f r o m p r i m a r y t o secondary w a l l f o r m a t i o n . F i g u r e 25 is taken f r o m such a phase i n the aplanospore o f V. ventricosa, a n d m a y correspond t o a n area i n w h i c h clusters o f T C ' s occur a n d move i n a d i r e c t i o n different f r o m the former o r i e n t a t i o n o f m i c r o f i b r i l s . L i n e a r T C ' s i n giant m a r i n e algae m a y be stable, because (a) t h e T C ' s d o n o t disappear f o l l o w i n g t r e a t m e n t w i t h c y c l o h e x i m i d e , a p r o t e i n synthesis i n h i b i t o r (34), a n d (b) g l u t a r a l d e h y d e t r e a t m e n t i n advance o f freeze fracture d i d n o t destroy the T C ' s ( u n p u b l i s h e d d a t a ) . T h u s , linear T C ' s i n giant m a r i n e algae m a y be involved i n the synthesis o f ordered m i c r o f i b r i l s w i t h m u c h longer lifetimes t h a n the rosettes (53). R e c e n t l y , more evidence for hélicoïdal arrangement o f cellulose m i crofibrils has been reported for a variety o f plant cells (54,55). I n Nitella, the hélicoïdal o r i e n t a t i o n o f cellulose m i c r o f i b r i l s was s h o w n t o arise by a m e c h a n i s m s i m i l a r t o self-assembly o f a cholesteric l i q u i d c r y s t a l (56). T h e c o n t r o l o f m i c r o f i b r i l o r i e n t a t i o n was p r i m a r i l y at the interface between t h e p l a s m a membrane a n d the innermost lamellae o f the n e w l y formed w a l l . T h e hélicoïdal w a l l , characterized b y a successive change o f cellulose m i crofibrils, was supposed t o be synthesized i n the i n n e r m o s t surface o f t h e new w a l l layer. I f t h i s is true, then t he involvement o f m i c r o t u b u l e s i n higher p l a n t cells is m u c h less probable t h a n p r e v i o u s l y believed. T h e selfassembly o f cellulose m i c r o f i b r i l s is related t o those cells w h i c h have such a p a t t e r n o f cell w a l l t h a t show arced o r i e n t a t i o n o f m i c r o f i b r i l s i n a t r a n s verse section (56). B o t h aplanospores a n d t h a l l u s cells o f B. forbesii have an arced p a t t e r n o f cellulose m i c r o f i b r i l s . However, we showed t h a t t h e m i c r o f i b r i l o r i e n t a t i o n i n b o t h spherical cells a n d e l o n g a t i n g r h i z o i d s was independent o f the o r i e n t a t i o n o f m i c r o t u b u l e s . W h i l e t h i s evidence does not c o n t r a d i c t the self-assembly m e c h a n i s m i n hélicoïdal walls o f green a l gae, b o t h hypotheses need a d d i t i o n a l e x p e r i m e n t a l v e r i f i c a t i o n . A cknowledgment s T h e a u t h o r t h a n k s D r . R . M a l c o l m B r o w n , J r . , at the U n i v e r s i t y o f T e x a s at A u s t i n , T e x a s , for the present p a r t o f the present s t u d y .

Literature Cited 1. Sugiyama, J.; Harada, H.; Fujiyoshi, Y.; Uyeda, N. Mokuzai Gakkaishi 1984, 30, 98-99. 2. Sugiyama, J.; Harada, H.; Fujiyoshi, Y.; Uyeda, N. Mokuzai Gakkaishi 1985, 31, 61-67. 3. Sugiyama, J.; Harada, H.; Fujiyoshi, Y.; Uyeda, N. Planta 1985, 166, 161-68.



276


POLYMERS

4. Kuga, S.; Brown, R. M., Jr. J. Electron Microscop. Tech. 1987, 6, 34956. 5. Kuga, S.; Brown, R. M., Jr. Polym. Commun. 1987, 28, 311-14. 6. Lin, F. C.; Cooper, J.; Delmer, D. P. Science 1985, 230, 822-25. 7. Brown, R. M., Jr.; Montezinos, D. Proc. Natl. Acad. Sci. USA 1976, 73, 143-47. 8. Giddings, T. H., Jr.; Brower, D. L.; Staehelin, L. A. J. Cell Biol. 1980, 84, 327-39. 9. Wada, M.; Staehelin, L. A. Planta 1981, 151, 462-68. 10. Reiss, H. Planta 1984, 160, 428-35. 11. Herth, W. In Botanical Microscopy; Rolands, A. W., Ed.; 1985, 285310. 12. Chafe, S. C. Wood Sci. Technol. 1978, 12, 203-17. 13. Mueller, S. C.; Brown, R. M., Jr. Planta 1982, 154, 489-500. 14. Roberts, K.; Burgess, J.; Roberts, I.; Linstead, P. In Botanical Mi croscopy; Robards, A. W., Ed.; 1985, 263-83. 15. Giddings, T. H., Jr.; Staehelin, L. A. Planta 173, 22-30. 16. Staehelin, L. Α.; Giddings, T. H., Jr. In Developmental Order: Its Ori gin and Regulation; Sabtelny, S., and Green, P. B., Eds.; Liss: New York, 1982; p. 133-47. 17. Itoh, T.; Brown, R. M., Jr. Planta 1984, 160, 372-81. 18. Quader and Robinson, Ber. Deutsch. Bot. Ges. 1981, 94, 75-84. 19. Brown, R. M., Jr. J. Cell Sci. Suppl. 1985, 2, 13-32. 20. Willison, J. H. M.; Brown, R. M., Jr. J. Cell Biol. 1978, 77, 103-19. 21. Itoh, T.; O'Neil, R. M.; Brown, R. M., Jr. Protoplasma 1984, 123, 174-83. 22. Mizuta, S. Plant Cell Physiol. 1985, 26, 53-62. 23. Mizuta, S. Plant Cell Physiol. 1985, 26, 1443-53. 24. Itoh, T. Proc. Intern. Dissolv. Pulps Conf. 1987, 117-20. 25. Mizuta, S.; Okuda, K. Botanica Marina 1987, 30, 205-15. 26. Hogetsu, T. Plant Cell Physiol. 1983, 24, 777-81. 27. Kiermayer, O.; Sleytr, U. B. Protoplasma 1979, 101, 133-38. 28. Noguchi, T.; Tanaka, K.; Ueda, K. Cell Struct. Fund. 1981, 6, 217-29. 29. Hotchkiss, A. T., personal communication. 30. Herth, W. Planta 1983, 159, 347-56. 31. Brown, R. M., Jr. The Third Philip Morris Science Symposium 1978, 52-123. 32. Hotchkiss, A. T.; Brown, R. M., Jr. J. Phycol. 1987, 23, 229-37. 33. Quader, H. J. Cell Sci. 1986, 83, 223-34. 34. Itoh, T.; Legge, R. L.; Brown, R. M., Jr. J. Phycol. 1986, 22, 224-32. 35. Steward, F. C.; Muhlethaler, K. Ann. Bot. N.S. 1953, 17, 295-316. 36. Cranshaw, J.; Preston, R. D. Proc. R. Soc. Β London Ser. 1958, 148, 137-48. 37. Mizuta, S.; Wada, S. Bot. Mag. Tokyo 1981, 94, 343-53. 38. Lloyd, C. W. Intern. Rev. Cytol. 1984, 86, 1-51. 39. Haigler, C. H. In Cellulose Chemistry and its Applications; Nevell, T. P.; Zeronian, S. H., Eds.; Ellis Horwood; 1987, p. 30-83.



19.

ITOH


277

40. Delmer, D. P. Adv. Carbohyd. Chem. & Biochem. 1983, 41, 105-53. 41. Hogetsu, T.; Takeuchi, Y. Planta 1982, 154, 426-34. 42. Franke, W. W.; Seib, E.; Osborn, M.; Weber, K.; Herth, W.; Falk, H. Cytobiol. 1977, 15, 24-48. 43. Lloyd, C. W. Nature 1979, 279, 239-41. 44. Lloyd, C. W. Ann. Rev. Plant Physiol. 1987, 38, 119-39. 45. Ishizawa, K.; Wada, S. Plant Cell Physiol. 1979, 20, 973-82. 46. Emons, A. M. E.; Wolters-Arts, A. M. C. Protoplasma 1983, 117, 6881. 47. Emons, A. M. C. Planta 1985, 163, 350-59. 48. Traas, J. Α.; Braat, P.; Emons, A. M. C.; Meeks, M.; Derksen, J. J. Cell Sci. 1985, 76, 303-20. 49. LaClaire, J. W., II. Planta 1987, 171, 30-42. 50. Mizuta, S.; Okuda, K. Bot. Gaz. 1987, 148, 297-307. 51. Okuda, Κ.; Mizuta, S. Plant Cell Physiol. 1987, 28, 461-73. 52. Itoh, T.; Brown, R. M., Jr. Protoplasma 1988, 144, 160-69. 53. Schneider, B.; Herth, W. Protoplasma 1986, 131, 142-52. 54. Vian, Β.; Roland, J. C. New Phytol. 1987, 105, 345-57. 55. Roland, J. C.; Reis, D.; Vian, B.; Satiat-Jeunemaitre, B.; Mosiniak, M. Protoplasma 1987, 140, 75-91. 56. Neville, A. C.; Levy, S. Planta 1984, 162, 370-84. RECEIVED March 27, 1989


Biogenesis of Cellulose Microfibrils and the Role of Microtubules in

Recommend Documents