Cellulose Biosynthesis - American Chemical Society

Chapter 17

Cellulose Biosynthesis

Downloaded by UNIV OF CALIFORNIA SAN DIEGO on March 12, 2017 | http://pubs.acs.org Publication Date: July 31, 1989 | doi: 10.1021/bk-1989-0399.ch017

The Terminal Complex Hypothesis and Its Relationship to Other Contemporary Research Topics Arland T. Hotchkiss, Jr. U.S. Department of Agriculture, Agricultural Research Service, Eastern Regional Research Center, 600 East Mermaid Lane, Philadelphia, PA 19118

Cellulose biosynthesis is a complex, sensitive, and not fully characterized process that occurs in organisms ranging from plants to bacteria to animals. Two fundamental approaches have been used to investigate cellulose biosynthesis; one structural and the other biochemical. The terminal complex hypothesis proposes that the cellulose synthesizing enzyme complex can be visualized with electron microscopy. Terminal complex is the name given to collections of plasma membrane particles thought to represent the cellulose synthase. While direct evidence is still not available to support this hypothesis, the amount of indirect supporting evidence has grown dramatically in the past few years. The relationship between terminal complexes, cellulose physical structure and the biochemical events of cellulose biosynthesis will be discussed. C e l l u l o s e , a p o l y s a c c h a r i d e c o n s i s t i n g o f linear l , 4 - / ? - D - a n h y d r o g l u c o p y r a nose chains l a t e r a l l y associated b y h y d r o g e n bonds, is t h e most a b u n d a n t a n d c o m m e r c i a l l y i m p o r t a n t p l a n t cell w a l l p o l y m e r (1). C o n s e q u e n t l y , cellulose is also one o f the most t h o r o u g h l y investigated p l a n t cell w a l l p o l y m e r s . However, i t is e n i g m a t i c i n the sense t h a t significant elements o f cellulose p h y s i c a l s t r u c t u r e a n d the m e c h a n i s m o f cellulose biosynthesis s t i l l are n o t w e l l u n d e r s t o o d . Since these subjects have been reviewed recently (2-10), this review w i l l u p d a t e topics covered p r e v i o u s l y a n d p r o v i d e a new analysis o f selected topics o f c o n t e m p o r a r y interest. Cellulose

Assembly

T h e t e r m i n a l c o m p l e x hypothesis proposes t h a t s t r u c t u r a l m a n i f e s t a t i o n s of the cellulose synthase e n z y m e c o m p l e x can be v i s u a l i z e d w i t h t h e freeze fracture s p e c i m e n p r e p a r a t i o n technique for electron microscopy. These This chapter not subject to U.S. copyright Published 1989 American Chemical Society

Lewis and Paice; Plant Cell Wall Polymers ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

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s t r u c t u r e s , c o n s i s t i n g o f collections of i n t r a m e m b r a n o u s p a r t i c l e s observed o n the i n t e r n a l f r a c t u r e faces o f the p l a s m a m e m b r a n e , are f r e q u e n t l y asso c i a t e d w i t h the ends of cellulose m i c r o f i b r i l i m p r e s s i o n s . F u r t h e r m o r e , since b i o c h e m i c a l evidence has d e m o n s t r a t e d t h a t cellulose biosynthesis occurs at the p l a s m a m e m b r a n e a n d t e r m i n a l complexes are l o c a t e d at the site of cellulose m i c r o f i b r i l assembly, the hypothesis proposes t h a t t e r m i n a l c o m plexes are the cellulose synthase p r o t e i n complexes. Since Roelofsen (11) a n d P r e s t o n (12) first p r o v i d e d the c o n c e p t u a l b a sis for the t e r m i n a l c o m p l e x h y p o t h e s i s , these s t r u c t u r e s have been r e p o r t e d i n a v a r i e t y o f o r g a n i s m s reviewed b y B r o w n (6). T h e r o s e t t e / g l o b u l e t e r m i n a l c o m p l e x consists o f a c o l l e c t i o n of s i x p a r t i c l e s a r r a n g e d h e x a g o n a l l y o n the p r o t o p l a s m i c face ( P F ) w i t h a c o m p l e m e n t a r y g l o b u l e o n the exo p l a s m i c face ( E F ) of the p l a s m a m e m b r a n e . Since the last review (6), rosette t e r m i n a l complexes have been observed i n the v a s c u l a r p l a n t s Lepidium sativum L . (13) a n d Zinnia elegans (14), as well as i n the green algae Chara globularis v a r . capillacta (15), Nitella translucens v a r . axillaris (16), a n d Mougeotia sp. (17,18). T h e linear t e r m i n a l c o m p l e x consists of p a r t i c l e rows (single, t r i p l e , or d i a g o n a l ) a n d have been r e p o r t e d o n the E F , P F , or b o t h faces of the p l a s m a m e m b r a n e . L i n e a r t e r m i n a l complexes c o n s i s t i n g of d i a g o n a l rows of P F i n t r a m e m b r a n o u s p a r t i c l e s have been r e p o r t e d re cently i n Vaucheria o f the X a n t h o p h y c e a e (19). T h e r e m a r k a b l e d i c h o t o m y between the t a x o n o m i c d i s t r i b u t i o n of the r o s e t t e / g l o b u l e a n d l i n e a r types of t e r m i n a l complexes continues to exist (18). R o s e t t e / g l o b u l e t e r m i n a l complexes have been observed t h r o u g h o u t the e v o l u t i o n a r y s p e c t r u m of o r g a n i s m s f r o m p r i m i t i v e p l a n t s s u c h as green algae ( C h a r o p h y c e a e ) to a d vanced vascular p l a n t s . H o w e v e r , linear t e r m i n a l complexes are o n l y f o u n d i n c e r t a i n a l g a l groups; the C h l o r o p h y c e a e a n d U l v o p h y c e a e classes i n the green algae, the X a n t h o p h y c e a e (yellow-green algae) a n d the P h a e o p h y c e a e ( b r o w n algae, Pelvetia, 20). D i r e c t m i c r o s c o p i c evidence d e m o n s t r a t i n g t h a t t e r m i n a l c o m p l e x p a r ticles are cellulose s y n t h e s i z i n g enzymes is not c u r r e n t l y available a n d w i l l await the p r o d u c t i o n of a n t i b o d i e s against cellulose s y n t h a s e f o l l o w i n g its i s o l a t i o n a n d p u r i f i c a t i o n . H o w e v e r , the p r o p o s a l t h a t t e r m i n a l complexes are p a r t of the cellulose synthase c o m p l e x is i n c r e a s i n g l y b e c o m i n g accepted (2) due to the a c c u m u l a t i o n of i n d i r e c t evidence s u p p o r t i n g t h i s h y p o t h e sis. S o m e of the most c o n v i n c i n g d a t a correlates h i g h densities of t e r m i n a l complexes w i t h l o c a l i z e d d e p o s i t i o n of cellulose m i c r o f i b r i l s d u r i n g c e r t a i n stages of p l a n t c e l l u l a r development. R o s e t t e / g l o b u l e t e r m i n a l c o m p l e x d e n s i t y values u p to 191 per μπι were observed u n d e r the s e c o n d a r y cell w a l l t h i c k e n i n g s of x y l e m t r a c h e a r y elements o f Lepidium sativum (13) a n d o f Zinnia elegans (14). It has been k n o w n for q u i t e some t i m e t h a t i n t i p - g r o w i n g p l a n t cells the d e n s i t y of r o s e t t e / g l o b u l e t e r m i n a l complexes increases d r a m a t i c a l l y at the t i p (up to 48 rosettes per / i m ) , where the most active cellulose m i c r o f i b r i l d e p o s i t i o n occurs (21,22). 2

2

T h e i d e n t i f i c a t i o n of t e r m i n a l complexes i n the G r a m - n e g a t i v e b a c t e r i u m Acetobacter xylinum n o w appears to be i n d o u b t . P r e v i o u s l y , a single linear r o w of p a r t i c l e s observed o n the outer l i p o p o l y s a c c h a r i d e m e m b r a n e



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P F h a d been proposed as the t e r m i n a l c o m p l e x (23) a n d associated pores were r e p o r t e d o n the outer m e m b r a n e E F (24). D u e to t h e i r p r o x i m i t y to the site o f cellulose r i b b o n e x t r u s i o n f r o m the cell surface, these s t r u c t u r e s were assumed t o be responsible for cellulose synthesis. A m o d e l was a d vanced i n w h i c h cellulose synthase was l o c a l i z e d o n the outer m e m b r a n e , w h i c h invoked adhesion sites between the outer a n d p l a s m a m e m b r a n e s as a m e c h a n i s m to e x p l a i n the transfer o f u r i d i n e - d i p h o s p h o r y l - g l u c o s e ( U D P G ) f r o m the c y t o p l a s m to the cellulose synthases (25,26). H o w e v e r , w h e n the outer a n d p l a s m a membranes of Acetobacier were i s o l a t e d separately b y d e n s i t y - g r a d i e n t c e n t r i f u g a t i o n , the cellulose synthase a c t i v i t y was l o c a l i z e d o n l y i n the p l a s m a m e m b r a n e f r a c t i o n (27). Therefore, the l i n e a r s t r u c t u r e s observed o n the Acetobacier outer m e m b r a n e , w h i l e they m a y be associated i n some m a n n e r w i t h cellulose biosynthesis, are p r o b a b l y not the cellulose synthase t e r m i n a l complexes. Since no u l t r a s t r u c t u r a l evidence for adhe sion sites between the outer a n d p l a s m a membranes has been presented, a t h o r o u g h i n v e s t i g a t i o n of the m e c h a n i s m of β (1-4) g l u c a n chain t r a n s l o c a t i o n f r o m the c y t o p l a s m i c m e m b r a n e to the outer m e m b r a n e i n Acetobacier xylinum is n o w i n order. Terminal Complex Structure and Phylogeny I n f o r m a t i o n derived f r o m t e r m i n a l c o m p l e x s t r u c t u r e has been used to probe phylogenetic r e l a t i o n s h i p s between cellulose p r o d u c i n g o r g a n i s m s (6,17,18,26,28-30). A s o r i g i n a l l y proposed (26), four characteristics o f cel lulose assembly (fixed vs. m o b i l e sites of cellulose biosynthesis, l i n e a r vs. rosette t e r m i n a l complexes, consolidated vs. u n c o n s o l i d a t e d t e r m i n a l c o m plexes, p l a s m a m e m b r a n e i n s e r t i o n of t e r m i n a l complexes) were considered significant w i t h regard to phylogenetic r e l a t i o n s h i p s . W h i l e most of these characteristics are s t i l l considered significant, their i m p o r t a n c e i n deter m i n i n g phylogenetic r e l a t i o n s h i p s has been r e i n t e r p r e t e d . C o n s e q u e n t l y , changes were m a d e i n the relative positions of o r g a n i s m s possessing t e r m i n a l complexes i n phylogenetic schemes w h i c h also reflect other u l t r a s t r u c t u r a l a n d b i o c h e m i c a l characteristics (18). Fixed vs. mobile sites of cellulose biosynthesis. T h e phylogenetic u t i l i t y of the fixed vs. m o b i l e site characteristic of cellulose biosynthesis reflects b a sic s t r u c t u r a l differences between p r o k a r y o t i c a n d e u k a r y o t i c o r g a n i s m s . In e u k a r y o t i c o r g a n i s m s , cellulose is p r o d u c e d f r o m t e r m i n a l complexes t h a t move i n the p l a n e of the " f l u i d - m o s a i c " p l a s m a m e m b r a n e b y the force generated f r o m m i c r o f i b r i l assembly (31), a n d deposit cellulose so t h a t i t envelops the cell. In c o n t r a s t , most c e l l u l o s e - p r o d u c i n g p r o k a r y o t i c o r g a n isms ( i n c l u d i n g Acetobacier, Achromobacier, Aerobacter, Agrobactenum, Alcaligenes, Azotobacter, Pseudomonas a n d Rhizobium; 32) e x t r u d e c e l l u lose as a r i b b o n l i k e e x t r a c e l l u l a r p r o d u c t f r o m a single fixed site o n the cell surface. However, a cellulosic e x t r a c e l l u l a r layer was r e p o r t e d i n Sarcina (33). It w o u l d not be possible for the m o b i l e site m e c h a n i s m o f cellulose biosynthesis to exist i n prokaryotes due to the c o m p l i c a t i o n of cellulose e x t r u s i o n t h r o u g h the p e p t i d y l g l y c a n cell w a l l a n d outer m e m b r a n e .



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Consolidation of rosette/globule terminal complexes. T h e strongly conserved n a t u r e of t e r m i n a l c o m p l e x m o r p h o l o g y i n c e r t a i n e u k a r y o t i c t a x o n o m i c groups led t o the r e o r g a n i z a t i o n o f phylogenetic r e l a t i o n s h i p s , w h i c h were based o n cellulose b i o s y n t h e s i s , a l o n g either a rosette t e r m i n a l c o m p l e x p a t h w a y or a l i n e a r t e r m i n a l c o m p l e x p a t h w a y (28). T h e significance of t e r m i n a l c o m p l e x c o n s o l i d a t i o n is most o b v i o u s w i t h z y g n e m a t a l e a n a l gae i n the rosette p a t h w a y . I n t h i s case, b o t h s o l i t a r y t e r m i n a l complexes a n d t e r m i n a l c o m p l e x rows are associated w i t h m i c r o f i b r i l a s s e m b l y d u r i n g p r i m a r y w a l l f o r m a t i o n , whereas h e x a g o n a l a r r a y s of t e r m i n a l complexes are i n v o l v e d i n secondary w a l l m i c r o f i b r i l assembly. T h e s e e x a m p l e s r e p resent three levels o f t e r m i n a l c o m p l e x c o n s o l i d a t i o n . F i r s t , the s o l i t a r y rosette consists o f s i x i n t r a m e m b r a n o u s p a r t i c l e s c o n s o l i d a t e d i n a p a t t e r n t h a t contains s i x - f o l d r o t a t i o n a l s y m m e t r y . S e c o n d - a n d t h i r d - o r d e r c o n s o l i d a t i o n is observed i n the l i n e a r t r a n s l a t i o n o f r o s e t t e / g l o b u l e t e r m i n a l complexes i n t o rows or h e x a g o n a l a r r a y s . T e r m i n a l c o m p l e x c o n s o l i d a t i o n has also been r e p o r t e d i n v a s c u l a r p l a n t s as loosely a l i g n e d files of rosettes associated w i t h secondary w a l l f o r m a t i o n (13,14,34,35). S i m i l a r rosette files were also observed d u r i n g p r i m a r y w a l l f o r m a t i o n i n r a p i d l y e l o n g a t i n g regions of Avena coleoptiles (6,36). W h e n coleoptiles were g r a v i s t i m u l a t e d , t e r m i n a l c o m p l e x d i s a g g r e g a t i o n o c c u r r e d o n l y o n the lower coleoptile h e m i c y l i n d e r as evidenced b y the o b s e r v a t i o n o f s o l i t a r y g l o b u l e t e r m i n a l complexes (6,36). It was p r o p o s e d t h a t s o l i t a r y t e r m i n a l complexes p r o d u c e d m i c r o f i b r i l s w i t h less i n t e r m i c r o f i b r i l l a r h y d r o g e n b o n d i n g t h a n was present between m i c r o f i b r i l s d e p o s i t e d b y c o n s o l i d a t e d t e r m i n a l complexes, a l l o w i n g the lower h e m i c y l i n d e r t o b e n d u p w a r d (36). T h e r e appears t o be a direct c o r r e l a t i o n between t e r m i n a l c o m p l e x l e n g t h (linear c o n s o l i d a t i o n ) a n d the w i d t h of the m i c r o f i b r i l p r o d u c e d . T h e best e x a m p l e of t h i s c o r r e l a t i o n is i n the d e p o s i t i o n of secondary w a l l m i c r o f i b r i l s i n Micrasterias b y h e x a g o n a l a r r a y s of rosettes (37). I n t h i s e x a m p l e , the longest row of rosettes (up t o 16 rosettes) l o c a t e d i n the center o f the a r r a y was associated w i t h the widest m i c r o f i b r i l s (up t o 28.5 n m ) , w h i l e shorter rows were associated w i t h narrower m i c r o f i b r i l s . T h e r e l a t i o n s h i p between the n u m b e r of rosettes i n a row a n d m i c r o f i b r i l w i d t h is not p r o p o r t i o n a l , however, since rows of 5 rosettes were associated w i t h the d e p o s i t i o n of 20 n m m i c r o f i b r i l s i n Spirogyra (38) a n d s o l i t a r y rosettes were associated w i t h 8 n m m i c r o f i b r i l s i n Mougeotia (17). T h e loosely associated files o f rosettes i n v o l v e d i n secondary w a l l f o r m a t i o n i n v a s c u l a r p l a n t s have less l i n e a r order t h a n the rosette rows f o u n d i n z y g n e m a t a l e a n h e x a g o n a l a r r a y s . C o r r e s p o n d i n g l y , the m i c r o f i b r i l w i d t h s o f the former were narrower t h a n those i n the l a t t e r . T h e consequence o f the t w o types of secondary w a l l f o r m a t i o n is t h a t the z y g n e m a t a l e a n secondary cell w a l l is m o r e r i g i d t h a n t h a t t y p i c a l o f vascular p l a n t s (30), a c h a r a c t e r i s t i c t h a t m a y be m u t u a l l y advantageous for each o r g a n i s m i n a f u n c t i o n a l sense. Consolidation of linear terminal complexes. T h e c o r r e l a t i o n between l i n e a r c o n s o l i d a t i o n o f t e r m i n a l complexes a n d m i c r o f i b r i l w i d t h does not appear to be as consistent for linear t e r m i n a l complexes, a l t h o u g h t h i s c o r r e l a t i o n



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was p r e v i o u s l y r e p o r t e d (2,39). Oocystis a n d Boergesenia h a d the same average t e r m i n a l c o m p l e x l e n g t h (510 n m ) , w h i c h was greater t h a n t h a t observed i n Valonia (350 n m ) a n d i n Vaucheria (192 n m ) , w h i l e the Oocystis, Valonia a n d Vaucheria m i c r o f i b r i l w i d t h s were s i m i l a r (20 n m ) b u t 10 n m less t h a n t h a t of Boergesenia (19,39,40). T h e s t r u c t u r a l developm e n t o f the linear t e r m i n a l c o m p l e x has recently been r e p o r t e d d u r i n g the regeneration o f Boergesenia a n d Valonia p r o t o p l a s t s f o l l o w i n g w o u n d i n g (41) . I n b o t h genera t e r m i n a l c o m p l e x linear c o n s o l i d a t i o n was r e p o r t e d t o increase d u r i n g p r i m a r y w a l l f o r m a t i o n , r e a c h i n g a m a x i m u m l e n g t h as secondary w a l l f o r m a t i o n c o m m e n c e d . S i m i l a r results also were observed i n Boodlea (Siphonocladales) d u r i n g p r i m a r y a n d secondary w a l l f o r m a t i o n (42) . T h u s , linear t e r m i n a l c o m p l e x c o n s o l i d a t i o n appears t o be a manifest a t i o n of the stage of cell w a l l development r a t h e r t h a n a significant factor i n the d e t e r m i n a t i o n of m i c r o f i b r i l d i m e n s i o n s . Plasma membrane insertion of terminal complexes. W h i l e the r o s e t t e / g l o b ule t e r m i n a l complexes observed i n the Z y g n e m a t a l e s (Micrastenas, Closterium, Spirogyra a n d Mougeotia) were p r e v i o u s l y t h o u g h t to be m o r e t r a n s m e m b r a n e t h a n those t y p i c a l of vascular p l a n t s (26,37), the former t e r m i n a l complexes are now considered to be more closely related t o the l a t t e r t h a n to the t r a n s m e m b r a n e linear t e r m i n a l complexes c h a r a c t e r i s t i c o f the U l v o p h y c e a e (18). T h i s statement does not i m p l y disagreement w i t h the o b s e r v a t i o n t h a t p a r t o f the rosette s t r u c t u r e m a y be p u l l e d away w i t h the globule w h e n the leaflets of the p l a s m a m e m b r a n e separate d u r i n g the freeze fracture process. Rosette s u b s t r u c t u r e appears to be a u n i v e r s a l characteristic o f g l o b u l a r t e r m i n a l complexes, since i t has been observed i n vascular p l a n t s (30,43) a n d i n the Z y g n e m a t a l e s (37). It now appears t h a t w h i l e the entire r o s e t t e / g l o b u l e t e r m i n a l c o m p l e x spans the p l a s m a m e m b r a n e based o n observations of c o m p l e m e n t a r y double replicas (37), neither the rosette nor the globule i n d i v i d u a l l y are t r a n s m e m b r a n e p a r t i cles as suggested p r e v i o u s l y (37). H e r t h (29) first r e p o r t e d t h a t the rosette t e r m i n a l c o m p l e x was c h a r a c teristic of those algae ( C h a r o p h y c e a e ) w h i c h represent the e v o l u t i o n a r y line t h a t gave rise to higher l a n d p l a n t s . Several other t a x o n o m i c characteristics also are t h o u g h t to s u p p o r t the p r o p o s a l t h a t the C h a r o p h y c e a e represents t h i s phylogenetic line (44). T h e r e f o r e , the i n s e r t i o n of the r o s e t t e / g l o b u l e t e r m i n a l c o m p l e x i n the p l a s m a m e m b r a n e does not appear to be p h y l o g e n e t i c a l l y significant, whereas t h i s is the case for the linear t e r m i n a l c o m p l e x . V a r i a t i o n i n linear t e r m i n a l c o m p l e x p l a s m a m e m b r a n e i n s e r t i o n exists i n o r g a n i s m s representing d i s t i n c t l y different t a x o n o m i c groups. O n l y E F l i n ear t e r m i n a l complexes are observed i n Oocystis ( C h l o r o p h y c e a e ) , w h i l e those i n Vaucheria ( X a n t h o p h y c e a e ) are observed o n l y o n the P F a n d those characteristic o f Valonia a n d Boergesenia ( U l v o p h y c e a e ) are f o u n d o n b o t h the E F a n d P F . T h e t a x o n o m i c groups represented b y those o r g a n i s m s possessing linear t e r m i n a l complexes are not considered to be closely r e l a t e d i n a phylogenetic sense based o n other u l t r a s t r u c t u r a l a n d b i o c h e m i c a l c h a r acteristics (44).


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Cellulose Structure V a r i a t i o n i n the p h y s i c a l s t r u c t u r e of cellulose has been observed a c c o r d i n g to its source a n d d e v e l o p m e n t a l stage (2,5). T h i s v a r i a t i o n , w h i c h i n c l u d e s differences i n m i c r o f i b r i l c r y s t a l l o g r a p h i c o r i e n t a t i o n , degree o f p o l y m e r i z a t i o n ( D P ) , transverse c r y s t a l l i n e d i m e n s i o n s ( c r y s t a l l i t e size), p a t t e r n s o f g l u c a n c h a i n h y d r o g e n b o n d i n g a n d g l u c a n c h a i n p o l a r i t y , has m a d e the basic c r y s t a l l i n e s t r u c t u r e o f cellulose difficult t o d e t e r m i n e . X - r a y diffract i o n studies have identified several c r y s t a l l i n e p o l y m o r p h s of cellulose (45). C e l l u l o s e isolated f r o m p l a n t s a n d b a c t e r i a t y p i c a l l y occurs i n the f o r m o f cellulose I (native cellulose). T h e cellulose II p o l y m o r p h is f o r m e d f r o m c e l lulose I b y t r e a t m e n t w i t h a l k a l i ( m e r c e r i z a t i o n ) or b y p r e c i p i t a t i o n f r o m s o l u t i o n . A reversal i n g l u c a n c h a i n p o l a r i t y f r o m p a r a l l e l to a n t i p a r a l l e l is t h o u g h t t o result f r o m the conversion f r o m cellulose I to I I . W h i l e cellulose I a n d II are the most c o m m o n p o l y m o r p h s , other f o r m s (cellulose I I I , I V a n d X ) have been r e p o r t e d (45). T h e cellulosic m i c r o f i b r i l s of Acetobacier, a n d those present i n the p r i m a r y a n d secondary walls of vascular p l a n t s are t w i s t e d a n d show no preferred c r y s t a l l o g r a p h i c o r i e n t a t i o n r e l a t i v e to the cell surface. H o w e v e r , flat c r y s t a l l o g r a p h i c a l l y o r i e n t e d m i c r o f i b r i l s are p r o d u c e d b y s i p h o n o c l a d a l e a n algae (Valonia, Boergesenia; U l v o p h y c e a e ; 46; R o b e r t s a n d H o t c h k i s s , u n p u b l i s h e d r e s u l t s ) , c l a d o p h o r a l e a n algae (Cladopkora, Chaetomorpha; Ulvophyceae; 47,48), z y g n e m a t a l e a n algae (Mougeotia; C h a r o p h y c e a e ; 18) a n d x a n t h o p h y c e a n algae (Vaucheria; 19) i n w h i c h the 6.OA l a t t i c e p l a n e o f cellulose is t y p i c a l l y p a r a l l e l to the cell surface ( u n i p l a n a r o r i e n t a t i o n , 1). T h e flat Spirogyra ( Z y g n e m a t a l e s ) m i c r o f i b r i l s appear to have u n u s u a l u n i p l a n a r o r i e n t a t i o n , since either the 3.9Â or the 5.4Â l a t t i c e planes have been r e p o r t e d t o p a r a l l e l the cell surface (49). U n u s u a l u n i p l a n a r o r i e n t a t i o n also has been r e p o r t e d i n Oedogonium ( C h l o r o p h y c e a e ) , i n w h i c h the 5.4Â l a t tice p l a n e was observed to p a r a l l e l the cell surface (1,50). Differences i n cellulose m i c r o f i b r i l c r y s t a l l o g r a p h i c o r i e n t a t i o n w i t h i n the Z y g n e m a t a l e s are t h o u g h t to result f r o m the presence (Spirogyra) or absence (Mougeotia) o f secondary w a l l f o r m a t i o n (18). I n t h i s r e g a r d , i t w i l l be i n t e r e s t i n g to see i f other z y g n e m a t a l e a n algae w i t h secondary w a l l f o r m a t i o n (i.e., Micrasterias, Closterium) possess the same u n u s u a l u n i p l a n a r o r i e n t a t i o n . F u r t h e r cellulosic s t r u c t u r a l v a r i a t i o n is d i s p l a y e d b y D P a n d c r y s t a l l i t e size p a r a m e t e r s . Acetobacier a n d vascular p l a n t p r i m a r y w a l l celluloses are low i n D P (2,000-6,000), w h i l e s i p h o n o c l a d a l e a n a n d v a s c u l a r p l a n t secondary w a l l celluloses are r e l a t i v e l y h i g h i n D P ( > 10,000) (2). D u r i n g c o t t o n fiber development, the cellulose I V p o l y m o r p h is p r o d u c e d d u r i n g p r i m a r y w a l l f o r m a t i o n , w h i l e i n secondary w a l l s , cellulose I is observed (51). T h e cellulose c r y s t a l l i t e size is highest i n u l v o p h y c e a n a n d c e r t a i n c h l o r o p h y c e a n algae (114-169Â), lowest i n vascular p l a n t s ( 4 9 - 6 2 Â ) a n d i n t e r m e d i a t e i n Acetobacier (70-84Â) (1). It appears t h a t cellulose c r y s t a l l i n e d i m e n s i o n s are i n d e p e n d e n t of the t y p e of t e r m i n a l cellulose s y n t h e s i z i n g c o m p l e x . T h e i d e a t h a t cellulose biosynthesis is not e x c l u s i v e l y responsible for d e t e r m i n i n g i t s c r y s t a l l i n e d i m e n s i o n s has been p r o p o s e d p r e v i o u s l y b y M a r x - F i g i n i (52).


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R a m a n spectroscopy a n d C C P - M A S N M R techniques have proved i m p o r t a n t i n the i n v e s t i g a t i o n o f cellulose c r y s t a l l i n e s t r u c t u r e (3). B a s e d o n the nonequivalence of alternate β (1-4) g l u c a n c h a i n g l y c o s i d i c linkages as d e t e r m i n e d b y R a m a n spectroscopy, i t was concluded t h a t the basic r e p e a t i n g u n i t was a disaccharide (53). T h e slight r i g h t a n d l e f t - h a n d e d d e v i a t i o n s f r o m a two-fold screw a x i s were a p p r o x i m a t e d b y those observed i n the c r y s t a l s t r u c t u r e s o f cellobiose a n d methyl-/?-cellobioside m o d e l d i saccharides. C e l l u l o s e c o m p u t e r models have also been generated based o n the g l y c o s i d i c oxygen b o n d r o t a t i o n a l angles. These c o m p u t e r models were r e p o r t e d t o better a p p r o x i m a t e the saddle p o s i t i o n between the t w o m a j o r r o t a t i o n a l angle energy o f conversion m i n i m a ( t h o u g h t to be r e p resentative o f n a t i v e cellulose) t h a n the cellobiose a n d methyl-/?-cellobiose c r y s t a l l o g r a p h i c models (54).


1 3

M i c r o s c o p i c evidence c o n f i r m i n g cellulose I g l u c a n c h a i n p o l a r i t y was r e p o r t e d p r e v i o u s l y w i t h Valonia cellulose (55,56). R e c e n t l y , p a r a l l e l c h a i n p o l a r i t y also was d e m o n s t r a t e d b y the a s y m m e t r i c a l a r r a n g e m e n t o f s i l v e r labeled r e d u c i n g ends at o n l y one end o f Acetobacier cellulose I fibrils (57). T w o d i s t i n c t c r y s t a l l i n e forms of cellulose I ( I a n d Ιβ) were r e p o r t e d b y A t a l l a a n d V a n d e r H a r t (58), based o n C P - M A S C N M R evidence. C e l lulose I a n d I^ differed o n l y i n t h e i r p a t t e r n s of hydrogen b o n d i n g , w h i l e their m o l e c u l a r c o n f o r m a t i o n s were otherwise i d e n t i c a l (3). A l l cellulose m i c r o f i b r i l s were t h o u g h t t o be m i x t u r e s o f I a n d lp forms w i t h the I f o r m p r e d o m i n a n t i n cellulose f r o m s i p h o n o c l a d a l e a n algae a n d Acetobac ier, whereas vascular p l a n t cellulose p r i m a r i l y consisted o f the \β f o r m . T h e s e conclusions were r e c e n t l y m o d i f i e d (59) f o l l o w i n g observations o f the preferential l s u s c e p t i b i l i t y to a c i d h y d r o l y s i s a n d m e c h a n i c a l b e a t i n g , as w e l l as s o l i d state C N M R m e t h o d s w h i c h enhance the c r y s t a l l i n e core resonances. It was d e t e r m i n e d t h a t vascular p l a n t cellulose consists almost e x c l u s i v e l y of the Ιβ f o r m , w i t h far less (if a n y ) I content t h a n r e p o r t e d earlier. T h e celluloses of Mougeotia a n d Chara were recently r e p o r t e d to be p r e d o m i n a n t l y 1^ (60). T h i s evidence suggests t h a t a c o r r e l a t i o n exists between the presence o f s o l i t a r y r o s e t t e / g l o b u l e t e r m i n a l cellulose s y n t h e s i z i n g complexes a n d the assembly of Ιβ cellulose (60). L i n e a r t e r m i n a l complexes m a y be associated w i t h the f o r m a t i o n of a m i x t u r e of b o t h I a n d Ιβ c r y s t a l l i n e forms. a

1 3

a

a

a

a

1 3

a

a

It now appears t h a t cellulose I is not e x c l u s i v e l y the n a t i v e p o l y m o r p h present i n a l l o r g a n i s m s . T h e results r e p o r t e d o r i g i n a l l y b y Sisson (61), w h i c h p r o v i d e d evidence t h a t cellulose II was the n a t i v e p o l y m o r p h present i n Halicystis ( U l v o p h y c e a e ) cell w a l l s , were recently reinvestigated a n d c o n firmed (62). A d d i t i o n a l l y , cellulose II p r o d u c i n g m u t a n t s o f Acetobacier have been isolated a n d a n a l y z e d w i t h x - r a y a n d low-dose electron diffrac t i o n (63). W h e n cellotetraose is i n d u c e d to c r y s t a l l i z e i n s o l u t i o n i t forms a s t r u c t u r e w h i c h has been used as a m o d e l c o m p o u n d a p p r o x i m a t i n g the c r y s t a l l o g r a p h i c n a t u r e of cellulose II based o n x - r a y d i f f r a c t i o n , electron diffraction a n d C P - M A S C N M R evidence (64). S i g n i f i c a n t l y , i n a l l cases where Acetobacier cellulose synthase in vitro a c t i v i t y has been r e p o r t e d , 1 3


17.

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the p r o d u c t was cellulose II as d e t e r m i n e d b y x - r a y a n d low-dose electron d i f f r a c t i o n (27,65). These observations i n d i c a t e t h a t cell-free synthesis of cellulose I is n o t k n o w n a n d t h a t the s p a t i a l a r r a n g e m e n t o f c o m p o n e n t s re s p o n s i b l e for the biosynthesis of cellulose I m a y be easily d i s t u r b e d . T h e r e fore, the biosynthesis of cellulose II i n n a t u r e m a y reflect a l t e r a t i o n s i n the s t r u c t u r e s responsible for cellulose assembly i n those o r g a n i s m s where i t has been observed.


In Vitro C e l l u l o s e S y n t h a s e A c t i v i t y W h i l e p a r t i a l p u r i f i c a t i o n o f U D P G : l , 4 - / ? - D - g l u c a n glucosyltransferase (cel lulose synthase) f r o m Acetobacter has been achieved (2,27,65), i t has not been possible t o d e m o n s t r a t e cellulose synthase a c t i v i t y i n s o l u b i l i z e d vas c u l a r p l a n t m e m b r a n e f r a c t i o n s . Instead, i s o l a t e d vascular p l a n t m e m b r a n e s p r o d u c e d β (1-3) g l u c a n (callose) u s i n g U D P G as a s u b s t r a t e . P r e v i o u s l y r e p o r t e d low levels o f β (1-4) g l u c a n in vitro synthesis i n vascular p l a n t s o l u b i l i z e d m e m b r a n e s are n o w t h o u g h t to represent x y l o g l u c a n b i o s y n t h e sis (2,66). A possible c a n d i d a t e for the Acetobacter cellulose s y n t h a s e has been p u r i f i e d as a n 83 K d c o n c a n a v a l i n Α-binding g l y c o p r o t e i n (65). A n earlier r e p o r t of in vitro cellulose biosynthesis b y Acetobacter d i g i t o n i n s o l u b i l i z e d membranes (67) is now considered to be i n d o u b t since the p r o d u c t f o r m e d was r e p o r t e d as cellulose I a n d a c o r r e l a t i o n between the in vitro p r o d u c t f o r m e d a n d the observed e l e c t r o n d i f f r a c t i o n p a t t e r n w a s n o t d e m o n s t r a t e d (27). C e l l u l o s e synthase a c t i v i t y was l o c a l i z e d o n the p l a s m a m e m b r a n e of Acetobacter (27), a n d is k n o w n to be r e g u l a t e d by b i s - f 3 ' 5 ' ) - c y c l i c d i g u a n y l i c a c i d , w h i c h is degraded b y a m e m b r a n e - b o u n d C a * sensitive phosphodiesterase (2,68). However, vascular p l a n t cellulose s y n thase does not appear to be under s i m i l a r c o n t r o l . A c c o r d i n g to D e l m e r (2), the vascular p l a n t cellulose synthase is a m u l t i f u n c t i o n a l / ? ( l - 3 ) : / ? ( l - 4 ) g l u cosyltransferase under the r e g u l a t i o n o f a n 18 K d 2 , 6 - d i c h l o r o b e n z o n i t r i l e ( D C B ) b i n d i n g p r o t e i n a n d C a . In order to test t h i s h y p o t h e s i s , a n t i bodies raised against the D C B b i n d i n g p r o t e i n a n d callose synthase w i l l be used to e x a m i n e t h e i r affinity for r o s e t t e / g l o b u l e t e r m i n a l complexes (69). F r o m the results of t h i s research, i t w i l l be possible to d e t e r m i n e whether cellulose synthase a n d callose synthase are the same e n z y m e complexes a n d i f the t e r m i n a l c o m p l e x s t r u c t u r e is a c t u a l l y associated w i t h cellulose biosynthesis. It also s h o u l d be noted t h a t N o r t h c o t e (70; see c h a p t e r 1, t h i s v o l ume) has proposed a m e c h a n i s m to e x p l a i n the p r o d u c t i o n of callose w h e n vascular p l a n t cells are d a m a g e d ( i n c l u d i n g d u r i n g m e m b r a n e i s o l a t i o n for in vitro cellulose synthase a c t i v i t y ) . I n t h i s m o d e l , the cellulose s y n t h a s e c o m p l e x includes a b i n d i n g p r o t e i n w h i c h c o n t r o l s the o r i e n t a t i o n o f the g r o w i n g g l u c a n c h a i n n o n - r e d u c i n g e n d . U n d e r n o r m a l c o n d i t i o n s , the C - 4 h y d r o x y l at the n o n - r e d u c i n g end is o r i e n t e d so t h a t i t is the most favored site for transfer of the next glucose f r o m U D P G . W h e n cell d a m a g e o c c u r s , the o r i e n t a t i o n o f the b i n d i n g p r o t e i n is d i s r u p t e d so t h a t the C - 3 h y d r o x y l is the m o s t favored site for acceptance o f glucose. However, t h i s m o d e l fails to e x p l a i n h o w the transfer of glucose to the g l u c a n c h a i n n o n - r e d u c i n g e n d +

2 +



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is c o o r d i n a t e d so t h a t each successive glucose is r o t a t e d 180° r e l a t i v e to the adjacent one. N o e x p e r i m e n t a l evidence is c u r r e n t l y available t o de t e r m i n e the possible c o r r e l a t i o n between the N o r t h c o t e m o d e l a n d electron m i c r o s c o p i c observations o f t e r m i n a l c o m p l e x s t r u c t u r e . Progress c o m p a r a b l e t o t h a t m a d e i n u n d e r s t a n d i n g the β (1-4) g l u c a n p o l y m e r i z a t i o n event has not been achieved w i t h the cellulose c r y s t a l l i z a t i o n process, since no evidence d e m o n s t r a t i n g the in vitro p r o d u c t i o n of cellulose I has been r e p o r t e d . H a i g l e r ' s cell-directed self-assembly m o d e l comes closest to e x p l a i n i n g cellulose c r y s t a l l i z a t i o n i n Acetobacter (25,26). T h i s m o d e l proposes t h a t linear rows of outer m e m b r a n e p a r t i c l e pores m a i n t a i n nascent β (1-4) g l u c a n chains i n 1.5 n m nondissociable fibrils as they are e x t r u d e d . O u t s i d e of the cell, g l u c a n chains i n register (established b y the outer m e m b r a n e pores) spontaneously self-assemble i n t o 3.5 n m cel lulose I m i c r o f i b r i l s . H o w the self-assembly of 1.5 n m fibrils occurs is the source of c u r rent debate. R u b e n a n d B o k e l m a n (71; see chapter, t h i s volume) have observed 1.78 n m s u b m i c r o f i b r i l s arranged i n a l e f t - h a n d - t w i s t e d , t r i p l e s t r a n d e d p a t t e r n w i t h i n 3.68 n m m i c r o f i b r i l s b y p l a t i n u m - c a r b o n s h a d o w i n g for electron microscopy. T h e y concluded t h a t t h i s t y p e of c o n s t r u c t i o n was i n c o m p a t i b l e w i t h the proposal (5,26) t h a t m i c r o f i b r i l s were f o r m e d b y the l a t e r a l fasciation of 1.5-1.8 n m fibrils a l o n g c r y s t a l l a t t i c e planes. A hélicoïdal association of s u b m i c r o f i b r i l s m e d i a t e d b y the h y d r o g e n - b o n d forces of x y l o s e - c o n t a i n i n g hemicellulosic polysaccharides was suggested as the m e c h a n i s m of self-assembly i n Acetobacter (71). However, the recent evidence t h a t c r y s t a l lattices of Acetobacter cellulose u p t o 25 n m w i d e were imaged b y electron m i c r o s c o p y (72), suggests t h a t c o n t i n u o u s , u n i n t e r r u p t e d c r y s t a l l i n e domains of cellulose exist, w h i c h i n t r i n s i c a l l y follow c r y s t a l l a t t i c e planes. F u r t h e r m o r e , no evidence for the presence of x y l o s e c o n t a i n i n g polysaccharides i n Acetobacter pellicles was confirmed ( G r e t z a n d H o t c h k i s s , u n p u b l i s h e d d a t a ) . Therefore, the self-assembly of 1.5 n m fibrils i n t o m i c r o f i b r i l s p r o b a b l y occurs i n a h e l i c a l f a s h i o n , b u t b y a m e c h a n i s m w h i c h m a i n t a i n s the s y m m e t r y of congruent l a t t i c e planes. M o r e evidence is needed to prove t h a t the self-assembly process is hélicoïdal. A l t e r a t i o n of Cellulose

Biosynthesis

A t t e m p t s to e x a m i n e the process of cellulose c r y s t a l l i z a t i o n have frequently i n v o l v e d c u l t u r i n g Acetobacter i n the presence of fluorescent brighteners, direct dyes, c a r b o x y - m e t h y l - c e l l u l o s e , or other agents w h i c h c o m p e t e for i n t e r c h a i n h y d r o g e n b o n d sites, thereby d i s r u p t i n g m i c r o f i b r i l f o r m a t i o n (5,26). T h e sheet-like s t r u c t u r e of the altered Acetobacter cellulose is now better u n d e r s t o o d following x - r a y a n d electron diffraction analysis (73). T h e results of t h i s s t u d y i n d i c a t e d t h a t fluorescent b r i g h t e n i n g agents, such as C a l c o f l u o r , stack transverse to the g l u c a n c h a i n l o n g axis a n d assume a h e l i c a l o r i e n t a t i o n due to g l u c a n c h a i n t w i s t i n g . T h e p r i m a r y forces i n v o l v e d i n the s t a c k i n g of dyes to the nascent g l u c a n chains were r e p o r t e d to be h y d r o p h o b i c interactions. H e l i c a l l y t w i s t e d fibrils of cellulose I c o u l d be regenerated f r o m the n o n c r y s t a l l i n e altered cellulose f o l l o w i n g water


17.

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w a s h i n g . However, a r e d u c t i o n i n c r y s t a l l i t e size relative t o t h a t t y p i c a l o f Acetobacter cellulose was observed. K a i (74) r e p o r t e d t h a t d y e - a l t e r e d cellulose was n o t t o t a l l y a m o r p h o u s , o b s e r v i n g reflections at 6.0Â. I n c o n t r a s t , the o n l y reflections H a i g l e r a n d C h a n z y (73) observed ( 3 . 9 9 Â ) w i t h s i m i l a r m a t e r i a l were a t t r i b u t e d t o the h e l i c a l s t a c k i n g o f the dye. T h e s e results suggest t h a t i f the events o f cell-directed e x t r u s i o n a n d nascent g l u c a n c h a i n self-assembly become u n c o u p l e d (as i n the case o f c e l lulose regeneration f r o m d y e - a l t e r e d cellulose), the n a t i v e c r y s t a l l i n e c e l l u lose d i m e n s i o n s w i l l not be achieved. T h e cell-directed self-assembly m o d e l is s t r e n g t h e n e d b y t h i s i n f o r m a t i o n , w i t h the c o r o l l a r y t h a t the cellulose synthase is l o c a l i z e d o n the p l a s m a m e m b r a n e as suggested b y B u r e a u a n d B r o w n (27). It appears obvious t h a t , regardless of h o w the nascent g l u c a n chains traverse the p e p t i d y l g l y c a n cell w a l l a n d outer m e m b r a n e , the c r i t i c a l s t r u c t u r a l c o m p o n e n t s responsible for c e l l - d i r e c t e d e x t r u s i o n are l o c a l i z e d o n the outer m e m b r a n e . A b s e n c e o f the l i n e a r r o w o f outer m e m b r a n e p a r t i c l e s i n cellulose II p r o d u c i n g m u t a n t s (63) suggests t h a t the c e l l directed e x t r u s i o n m e c h a n i s m was a l t e r e d , l e a d i n g to the c r y s t a l l i z a t i o n o f cellulose I I . F u t u r e research e x a m i n i n g the self-assembly of e x t r u d e d β (1-4) g l u c a n chains i n the presence of c o m p o u n d s t h a t undergo cholesteric l i q u i d c r y s t a l f o r m a t i o n s h o u l d also y i e l d v a l u a b l e i n f o r m a t i o n t h a t w i l l address the p o s s i b i l i t y of a hélicoïdal m e c h a n i s m for m i c r o f i b r i l c o n s t r u c t i o n . Since the events o f g l u c a n c h a i n p o l y m e r i z a t i o n a n d cellulose c r y s t a l l i z a t i o n are not s p a t i a l l y s e p a r a t e d i n e u k a r y o t i c o r g a n i s m s as t h e y are i n p r o k a r y o t i c o r g a n i s m s , the o b s e r v a t i o n of cellulose II i n the former (62,63) raises i n t e r e s t i n g questions c o n c e r n i n g the s t r u c t u r e of a t e r m i n a l c o m p l e x t h a t c o u l d assemble a n t i p a r a l l e l cellulose. O n e p o s s i b i l i t y is t h a t the P F c o m p o n e n t c o n t a i n s the cellulose synthase a c t i v i t y a n d the E F c o m p o n e n t either is m i s s i n g or lacks the a b i l i t y to a l i g n nascent g l u c a n c h a i n s i n a p a r a l l e l o r i e n t a t i o n . A l t e r n a t i v e l y , i f cellulose II c h a i n p o l a r i t y is p a r a l lel i n s t e a d of a n t i p a r a l l e l , t e r m i n a l c o m p l e x m e d i a t e d cellulose II a s s e m b l y w o u l d be m u c h m o r e easily e x p l a i n e d based o n o u r present knowledge. A p a r a l l e l c r y s t a l s t r u c t u r e m o d e l for cellulose I I has been described recently (75). It is i n t e r e s t i n g to consider the effects of n o n - c e l l u l o s i c cell w a l l p o l y s a c charides o n cellulose c r y s t a l l i z a t i o n i n e u k a r y o t i c o r g a n i s m s . T h e a d d i t i o n of p u r i f i e d p e a x y l o g l u c a n (76) or m a n n o d e x t r i n s ( A t a l l a , p e r s o n a l c o m m u n i c a t i o n ) to Acetobacter cultures has been r e p o r t e d to prevent or alter c e l lulose m i c r o f i b r i l c r y s t a l l i z a t i o n . These results suggest t h a t p l a n t cell w a l l p o l y s a c c h a r i d e s present d u r i n g m i c r o f i b r i l d e p o s i t i o n m a y alter cellulose biosynthesis. B a s e d o n ^ - N M R evidence (second m o m e n t a n d s o l i d echo a n a l y s i s ) , a m o d e l was proposed w h i c h suggests t h a t cellulose m i c r o f i b r i l s f o r m a h i g h l y o r d e r e d c o m p l e x w i t h a n i m m o b i l e - p o p u l a t i o n of x y l o g l u c a n i n the p r i m a r y cell walls o f bean h y p o c o t y l s (77). W h i l e t h i s m o d e l is prel i m i n a r y , i t appears to i m p l y t h a t the i n t i m a t e association w i t h x y l o g l u c a n m a y be due t o c r y s t a l l i z a t i o n of cellulose i n the presence o f x y l o g l u c a n at the p l a s m a m e m b r a n e . These proposals represent a possible e x p l a n a t i o n for the observed v a r i a t i o n i n cellulose c r y s t a l l i t e sizes t h a t w o u l d be open to d e v e l o p m e n t a l r e g u l a t i o n based o n changes i n cell w a l l c o m p o s i t i o n .



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C e l l w a l l c o m p o n e n t s also have been r e p o r t e d to influence the p a t tern o f m i c r o f i b r i l d e p o s i t i o n . G l u c u r o n o x y l a n has been affinity-labeled ( x y l a n a s e - g o l d c o m p l e x ) preferentially at the p o i n t s of hélicoïdal m i c r o f i b r i l o r i e n t a t i o n shifts between S i a n d S2 lamellae i n Tilia plaiyphyllos wood (78). It was proposed t h a t the s t r u c t u r a l a t t r i b u t e s o f the h e m i c e l l u l o s i c g l u c u r o n o x y l a n (elongated stiff backbone w i t h short, flexible side chains) were favorable for cholesteric l i q u i d c r y s t a l f o r m a t i o n . Therefore, t h r o u g h close a s s o c i a t i o n w i t h the cellulose, the g l u c u r o n o x y l a n c o u l d i n d u c e a hélicoïdal t r a n s i t i o n between S i a n d S2 m i c r o f i b r i l o r i e n t a t i o n s . T h e t u r n o v e r of x y l o g l u c a n i n dicots a n d /?-glucan ( m i x e d 1-3, 1-4 linkages) i n m o n o cots c a t a l y z e d b y specific cell w a l l l o c a l i z e d glucanohydrolyases i n a d d i t i o n to C a / H + i o n exchange b y a c i d i c cell w a l l c a r b o h y d r a t e s , are t h o u g h t t o a l l o w the slippage r e o r i e n t a t i o n of cellulose m i c r o f i b r i l s to o c c u r d u r i n g cell e l o n g a t i o n (79). C e l l w a l l glycoproteins m a y also influence the s p a t i a l arrangement o f cellulose m i c r o f i b r i l s . T h e d i s t r i b u t i o n o f i s o d i t y rosine cross-links between adjacent h y d r o x y p r o l i n e - r i c h g l y c o p r o t e i n s has been proposed to establish a cell w a l l m a t r i x mesh w h i c h restricts the dep o s i t i o n of cellulose m i c r o f i b r i l s i n vascular plants (80). 2 +

Molecular Genetics of Cellulose Biosynthesis B i o c h e m i c a l knowledge of b i o s y n t h e t i c m e t a b o l i s m has often been a i d e d b y genetic studies of m u t a n t s defective i n key enzymes i n various a n a b o l i c p a t h w a y s . It is hoped t h a t t h i s research a p p r o a c h w i l l also be h e l p f u l i n investigations o f cellulose biosynthesis. C u r r e n t l y Acetobacier m u t a n t s deficient i n cellulosic pellicle p r o d u c t i o n (Pel"") have been p r o d u c e d (81,82). H o w e v e r , the P e l " m u t a n t s p r o d u c e d i n one s t u d y (82) s t i l l m a d e s m a l l quantities of cellulose II in vivo, possessed n o r m a l U D P G : l , 4 - / ? - D - g l u c a n g l u c o s y t r a n s f e r a s e a c t i v i t y in vitro a n d h a d no detectable galactose i n l i p o p o l y s a c c h a r i d e s ( L P S ) . These observations were i n t e r p r e t e d to m e a n t h a t the P e l " " defect was not i n the cellulose p o l y m e r i z a t i o n event b u t t h a t the defect m a y have been i n the p r e c e d i n g step c a t a l y z e d b y U D P G p y r o p h o s p h o r y l a s e (83). However, a n a d d i t i o n a l m u t a t i o n affecting the s t r u c t u r e s responsible for nascent g l u c a n c h a i n t r a n s l o c a t i o n t h r o u g h the p e p t i d y l g l u c a n a n d outer m e m b r a n e m a y also be present since cellulose II is p r o d u c e d i n vivo. T h e a l t e r a t i o n i n l i p o p o l y s a c c h a r i d e s t r u c t u r e m a y be a m a n i f e s t a t i o n o f the l a t t e r t y p e of defect i n P e l ~ m u t a n t s . F u r t h e r i n v e s t i g a t i o n o f these m u t a n t s s h o u l d p r o v i d e v a l u a b l e i n f o r m a t i o n a b o u t the m e c h a n i s m of cellulose II f o r m a t i o n a n d g l u c a n c h a i n t r a n s l o c a t i o n i n Acetobacter. I s o l a t i o n a n d sequencing of the cellulose synthase gene(s) has not been a c c o m p l i s h e d yet; however, D N A f r o m Acetobacter xylinum c o n t a i n i n g t h i s gene(s) was cloned i n t o b r o a d host-range p l a s m i d vectors (82). T h e s e vectors were m o b i l i z e d i n t o P e l " m u t a n t s to test for c o m p l e m e n t a t i o n . T o date, t h i s a p p r o a c h has not p r o d u c e d a p e l l i c l e - f o r m i n g t r a n s c o n j u g a n t f r o m a P e l " m u t a n t o f Acetobacter (82). T h e direct c o r r e l a t i o n between cellulose p r o d u c t i o n a n d presence o f p l a s m i d D N A i n Acetobacter has been r e p o r t e d


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(84), suggesting t h a t the cellulose synthase gene(s) was l o c a l i z e d o n one or m o r e p l a s m i d s . H o w e v e r , some Acetobacter s t r a i n s l a c k i n g p l a s m i d s or c u r e d o f p l a s m i d s were recently r e p o r t e d t o p r o d u c e cellulose (82). T h e r e fore, p l a s m i d s cannot be regarded as the exclusive site for cellulose s y n t h a s e genes.


Conclusions M u c h progress has been m a d e i n the field o f cellulose b i o s y n t h e s i s i n t h e past few years. T h e d i s t r i b u t i o n o f t e r m i n a l complexes i n p l a n t s h a s been m o r e f u l l y d e s c r i b e d . T h e g a p between u l t r a s t r u c t u r a l observations o f t e r m i n a l complexes a n d b i o c h e m i c a l evidence for t h e i r f u n c t i o n i n cellulose biosynthesis has been n a r r o w e d , l e a d i n g t o a g r o w i n g acceptance o f the t e r m i n a l c o m p l e x hypothesis i n t h e scientific c o m m u n i t y . I n Acetobacter, t h e cellulose synthase has been l o c a l i z e d o n t h e p l a s m a m e m b r a n e a n d s i g n i f i cant progress has been m a d e t o w a r d i t s i s o l a t i o n . H i g h r e s o l u t i o n evidence has been presented t o describe t h e process o f cell-directed self-assembly o f Acetobacter cellulose r i b b o n s . F u t u r e e x a m i n a t i o n o f the role o f cholesteric l i q u i d c r y s t a l l i z a t i o n i n cell-directed self-assembly m a y help t o resolve d i f ferences between t h i s m o d e l a n d the t r i p l e - s t r a n d e d , l e f t - h a n d - t w i s t e d c e l l u lose m i c r o f i b r i l m o d e l for cellulose c r y s t a l l i z a t i o n . T h e d i v e r s i t y o f cellulose p h y s i c a l s t r u c t u r e i n n a t u r e has been f u r t h e r defined. E s p e c i a l l y s i g n i f i c a n t i n t h i s regard have been t h e observations o f the c o r r e l a t i o n o f t e r m i n a l c o m p l e x t y p e w i t h cellulose I a n d 1^ s t r u c t u r e a n d the occurrence o f n a t i v e cellulose I I . D u e t o t h e a b u n d a n c e o f l i t e r a t u r e c o n c e r n i n g cellulose s t r u c t u r e a n d biogenesis, t h i s review was n o t i n t e n d e d t o be comprehensive i n n a t u r e . I n s t e a d , i n t e r p r e t a t i o n , s p e c u l a t i o n a n d a n a l y s i s o f recent progress i n v a r i ous areas o f cellulose biosynthesis research have been offered i n a n a t t e m p t to s t i m u l a t e new ideas a n d discussion. M a n y o f t h e recent i n v e s t i g a t i o n s e n u m e r a t e d c a n p o t e n t i a l l y m a k e significant c o n t r i b u t i o n s t o w a r d a b e t t e r u n d e r s t a n d i n g o f cellulose s t r u c t u r e a n d i t s biosynthesis i n t h e f u t u r e . T h e a u t h o r agrees w i t h D e l m e r (2,85) t h a t there is a m p l e o p p o r t u n i t y for n e w c o n t r i b u t o r s a n d novel approaches i n t h i s e n i g m a t i c field. a

A cknowledgment s I w o u l d like t o t h a n k E r i c R o b e r t s , R . M a l c o l m B r o w n , J r . , K e v i n H i c k s a n d J u l i a G o p l e r u d for m a n y h e l p f u l suggestions d u r i n g t h e p r e p a r a t i o n o f this manuscript. Literature Cited

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Cellulose Biosynthesis - American Chemical Society

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