Triple-Stranded Left-Hand Helical Cellulose Microfibril in Acetobacter

Chapter 20

Triple-Stranded Left-Hand Helical Cellulose Microfibril in Acetobacter xylinum and in Tobacco Primary Cell Wall Downloaded by UNIV OF CALIFORNIA SAN DIEGO on September 16, 2014 | http://pubs.acs.org Publication Date: July 31, 1989 | doi: 10.1021/bk-1989-0399.ch020

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George C. Ruben , Gordon H. Bokelman , and William Krakow 1

Department of Biological Sciences, Dartmouth College, Hanover, NH 03755 Philip Morris Research Center, P.O. Box 26583, Richmond, VA 23261 IBM, T. J. Watson Research Center, Box 218, Yorktown Heights, NY 10598

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Tobacco primary cell wall and normal bacterial Acetobacter xylinum cellulose formation produced a 36.8 ± 3Å triple-stranded left-hand helical microfibril in freeze-dried Pt-C replicas and in negatively stained preparations for transmission electron microscopy (TEM). A. xylinum growth in the presence of 0.25 mM Tinopal disrupted cellulose microfibril formation and produced a 17.8 ± 2.2Å left-hand helical submicrofibril. Models of the triple-stranded left-hand helical microfibril and the left-hand helical submicrofibril were directly compared to TEM images. Computer generated optical diffraction patterns of the models and the images were complex and similar. The submicrofibril appears to have the dimensions of a nine (1-4)-β-D-glucan parallel chain crystalline unit whose long, 23Å, and short, 19Å, diagonals form major and minor left-handed axial surface ridges every 36Å. Synthesis of the left-hand helical submicrofibril appears to be the driving force for self-assembly of a left-hand helical microfibril from three submicrofibrils. T h e g r a m negative b a c t e r i u m Acetobacier xylinum produces a r i b b o n of c r y s t a l l i n e cellulose I whose n e u t r a l sugar content is 9 6 . 8 % glucose a n d 3 . 2 % xylose (1). G r o w t h o f A. xylinum i n a m e d i u m c o n t a i n i n g 4,4-bis(4-anilino-6-bis (2-hydroxyethyl) amino-1,3,5-triazin-2-ylamino) 2 , 2 - stilbene-disulfonic a c i d , m a r k e t e d under c o m m o n names C a l c o f l u o r W h i t e S T or T i n o p a l L P W , c a n reversibly d i s r u p t n o r m a l r i b b o n f o r m a t i o n i n concentrations greater t h a n 0.1 m M a n d c a n increase the rate o f cellulose synthesis u p t o four times i n concentrations 1 m M o r greater (2-5). T h i s c o m p o u n d s t o i c h i o m e t r i c a l l y b i n d s t o glucose residues o f n e w l y p o l y m e r i z e d g l u c a n chains a n d makes cellulose I c r y s t a l l i n i t y i n the w e t state, measured 0097-6156/89/0399-0278$06.00/0 © 1989 American Chemical Society

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

Downloaded by UNIV OF CALIFORNIA SAN DIEGO on September 16, 2014 | http://pubs.acs.org Publication Date: July 31, 1989 | doi: 10.1021/bk-1989-0399.ch020

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b y X - r a y d i f f r a c t i o n , u n d e t e c t a b l e . I n the d r y state cellulose I c r y s t a l l i n i t y is present a n d , d e p e n d i n g o n C a l c o f l u o r c o n c e n t r a t i o n , the c r y s t a l l i t e sizes c a n be reduced f r o m 65 a n d 74À to 28Â ( 2 , 3 ) . Since m i c r o f i b r i l s are ass u m e d to c r y s t a l l i z e f r o m s m a l l filaments to larger ones b y l a t e r a l f a s c i a t i o n , t h i s enhanced g r o w t h rate a n d a n undetectable wet state c r y s t a l l i n i t y have been i n t e r p r e t e d as the s e p a r a t i o n of the p r i m a r y cellulose I p o l y m e r i z a t i o n step f r o m a s e q u e n t i a l c r y s t a l l i z a t i o n step (2-5) w h i c h is b l o c k e d . W e have i n v e s t i g a t e d A. xylinum cellulose p r o d u c t i o n i n the absence a n d presence o f 0.25 m M T i n o p a l a n d r e p o r t o n the freeze-dried s t r u c t u r e of the 3 6 . 8 ± 3 Â m i c r o f i b r i l p r o d u c e d under n o r m a l c o n d i t i o n s a n d the 1 7 . 8 ± 2 . 2 Â s u b m i c r o f i b r i l p r o d u c e d i n the presence o f T i n o p a l (1). W e have also f o u n d s u b m i c r o f i b r i l s a n d m i c r o f i b r i l s i n tobacco p r i m a r y cell w a l l s i m i l a r t o A. xylinum. W e present models of the t r i p l e - s t r a n d e d l e f t - h a n d h e l i c a l m i c r o f i b r i l , the l e f t - h a n d h e l i c a l s u b m i c r o f i b r i l a n d the a p p a r e n t r e l a t i o n s h i p of the four sugar c h a i n fiber d i f f r a c t i o n u n i t cell to the s u b m i c r o f i b r i l ( 1 , 6 ) . C o m p u t e r generated single molecule o p t i c a l d i f f r a c t i o n p a t t e r n s o f these models a n d of representative T E M m i c r o g r a p h s reinforce o u r i m p r e s s i o n o f congruency. T h e p a t t e r n s suggest t h a t the 17.8Â cellulose s u b m i c r o f i b r i l generated i n the presence of 0.25 m M T i n o p a l is o r g a n i z e d as a fibrillar u n i t w i t h n i n e p a r a l l e l sugar chains f o r m i n g a l e f t - h a n d e d h e l i c a l s t r u c t u r e (1). T h e prevalent a s s u m p t i o n t h a t the h i g h rate o f cellulose synthesis i n d u c e d i n A. xylinum b y T i n o p a l or C a l c o f l u o r is due to a cellulose p o l y m e r i z a t i o n step u n c o u p l e d f r o m a s e q u e n t i a l r a t e - l i m i t i n g c r y s t a l l i z a t i o n step is not consistent w i t h a n ordered c r y s t a l - l i k e s u b m i c r o f i b r i l . Methods and Materials T h e A. xylinum ( A m e r i c a n T y p e C u l t u r e C o l l e c t i o n 23769) was g r o w n o n 40 m M D-glucose a n d 0.5 M p h o s p h a t e ( p H 7, 2 0 ° C ) u n t i l i t f o r m e d a w h i t e , flocculent surface cap o n the s o l u t i o n . S a m p l e s p r e p a r e d i n t h i s w a y were t h e n g r o w n consecutively o n 0.25 m M T i n o p a l for 1 h , o n 0.25 m M T i n o p a l for 1.5 h , o n .025 m M T i n o p a l for 1 h , a n d t h e n o n 4 0 m M D glucose a n d 0.5 M p h o s p h a t e ( p H 7, 2 0 ° C ) for 1 h . E a c h 1.25 c m p e l l i c l e o f cellulose r i b b o n s w i t h cells g r o w i n g a n d tethered b y t h e i r r i b b o n s at i t s p e r i p h e r y was r i n s e d sequentially, first i n 5 separate dishes of w a t e r , t h e n i n 5 separate dishes of 1:3 e t h a n o l - w a t e r . E a c h p e l l i c l e was t h e n p l a c e d o n a 1.25 c m W h a t m a n 50 filter p a p e r disc, b l o t t e d , a n d frozen i n l i q u i d p r o p a n e . P e l l i c l e f r o m A. xylinum g r o w n n o r m a l l y was freeze-dried for 1.5 h at - 7 8 ° C , t h e n r e p l i c a t e d w i t h 17.3À P t - C (at - 1 7 8 ° C ) , a n d b a c k e d w i t h 90.2Â c a r b o n o n a W i l t e k Industries m o d i f i e d B a l z e r ' s 301 w i t h c r y o p u m p a n d r e b u i l t c o l d stage (7). T h e T i n o p a l - t r e a t e d s a m p l e was freeze-dried for 2.8 h at - 7 0 ° C , r e p l i c a t e d w i t h 16.4Â P t - C (at - 1 7 8 ° C ) , a n d backed w i t h 156Â o f c a r b o n . T h e t o b a c c o lower e p i d e r m a l peels were p r e p a r e d f r o m a C o k e r 319 leaf ( N o . 1 3 o n s t a l k ) . T h e s e peels were i m m e r s e d i n 1:3 e t h a n o l - w a t e r , b l o t t e d t o remove the excess s o l u t i o n a n d t h e n frozen o n 1.25 c m m i c a discs b y r a p i d i m m e r s i o n i n l i q u i d p r o p a n e ( - 1 9 0 ° C ) . T h i s s a m p l e was freeze-dried for 3 h at - 7 0 ° C , a n d t h e n r e p l i c a t e d w i t h 15.9Â P t - C (45° angle) at - 1 7 8 ° C in vacuo ( 6 . 6 7 / i P a ) a n d b a c k e d w i t h 139Â of f


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c a r b o n . A l l of the samples were digested i n 8 0 % s u l f u r i c a c i d . T h e replicas were r i n s e d i n deionized water a n d t h e n picked u p w i t h c a r b o n - c o a t e d , 300 m e s h grids f r o m u n d e r n e a t h a n d e x a m i n e d o n a J E M 1 0 0 C X i n s t r u m e n t as described p r e v i o u s l y (8). I n d i r e c t l y evaporated c a r b o n films of ~ 80Â thickness, suspended o n 300-mesh g r i d s , were used t o s u p p o r t A. xylinum cellulose t h a t h a d been treated w i t h b o i l i n g trifluoroacetic a c i d to remove hemicellulose. T h i s s a m p l e was negatively s t a i n e d w i t h 2 % u r a n y l acetate at p H 3.8, as p r e v i o u s l y described (9). T o contrast-enhance u n i d i r e c t i o n a l 15-18Â t h i c k P t - C - c o a t e d c e l l u lose specimens backed w i t h 100-173Â t h i c k c a r b o n films, m i c r o g r a p h s were contrast-reversed o n K o d a k 7302 fine-grain, p o s i t i v e film (8). I n a d d i t i o n to i n c r e a s i n g the contrast of 10-20Â features, the P t - C coated surfaces b e c a m e w h i t e , a n d the m o l e c u l a r details were m o d u l a t e d o n t h i s b a c k g r o u n d i n b l a c k s a n d shades of grey for easy s t r u c t u r a l i n t e r p r e t a t i o n (10-14). S h o o t i n g a t i l t series at 10° intervals at 1 0 x m a g n i f i c a t i o n o n a J E M 1 0 0 C X at 80 k V w i t h a 5 m m focal l e n g t h a n d a 40μπι objective a p e r t u r e achieved a 6.6Â r e s o l u t i o n a n d a 2 6 2 5 A d e p t h of field i n the p i c t u r e series (8). T h e t i l t series was generally viewed stereoscopically, a n d t h e n a single i m a g e representing the 3 - D s t r u c t u r e was s h o w n . I n order to e s t i m a t e the r e a l size of a filament u n d e r n e a t h its P t - C c o a t i n g ( u n i d i r e c t i o n a l at a 45° a n gle), the l o n g i t u d i n a l axis of a filament h a d to be w i t h i n 10° of the general s h a d o w - d i r e c t i o n o n the r e p l i c a surface, so t h a t b o t h sides of the filament were P t - C coated. T h e filament s h o u l d be r o u g h l y at a 45° angle w i t h the P t - C source (checked b y stereo-viewing), a l t h o u g h filaments w h i c h were P t - C coated at a p p r o x i m a t e l y a 90° angle were o n l y 1Â s m a l l e r (12). Series of fiber w i d t h measurements, m a d e at i m a g e m a g n i f i c a t i o n s of 2 t o 5 m i l l i o n , a n d u s u a l l y n u m b e r i n g fewer t h a n 100, were averaged, a n d the P t - C film thickness, measured o n the q u a r t z - c r y s t a l m o n i t o r , s u b t r a c t e d f r o m the average w i d t h , to give a n estimate of the real filament d i a m e t e r (11). It was recently f o u n d t h a t t h i s w i d t h - c o r r e c t i o n m e t h o d s h o u l d be reduced b y 1.5Â ( 1 2 , 1 3 ) . I n contrast, the center to center distance between either ridges or grooves a l o n g a P t - C coated m i c r o f i b r i l or s u b m i c r o f i b r i l were assumed to be u n c h a n g e d . 5

C o m p u t e r generated o p t i c a l diffraction p a t t e r n s of single cellulose m i crofibrils a n d s u b m i c r o f i b r i l s were o b t a i n e d f r o m large field m i c r o g r a p h s p r i n t e d at 1 0 x m a g n i f i c a t i o n . T h e images were d i g i t i z e d v i a a t e l e v i s i o n c a m e r a connected to a n image frame store a n d c o n t r o l l e d t h r o u g h a n I B M 3 0 9 D m a i n frame c o m p u t e r ( 1 5 , 1 6 ) . Briefly, a n area of the image c o n t a i n i n g a single molecule was selected b y a c i r c u l a r electronic a p e r t u r e defined b y a graphics overlay cursor under o p e r a t o r c o n t r o l . W h e n the image was s h i p p e d t o the host C P U , the fast fourier t r a n s f o r m ( F F T ) a n d subsequent power s p e c t r u m were c o m p u t e d f r o m the region defined e l e c t r o n i c a l l y , w i t h a n edge g r a d i n g f u n c t i o n to e l i m i n a t e h a r d edge d i f f r a c t i o n . T h e e l e c t r o n i c a l l y s a m p l e d i m a g e region a n d i t s power s p e c t r u m ( o p t i c a l d i f f r a c t i o n p a t t e r n ) were t h e n sent to a h a r d copy s l i d e - m a k i n g device. T h e diffract i o n p a t t e r n was c a l i b r a t e d u s i n g Keuffel & Esser (46 1513) 10 x 10 to c m g r a p h paper w i t h the same setup for d i g i t i z i n g the molecule. T h e o p t i c a l 6



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d i f f r a c t i o n p a t t e r n s were o r i e n t e d i n the same d i r e c t i o n as the spacings a p p e a r e d i n the molecule ( F i g u r e s 5, 6,7 a n d 10) a n d were generally scaled over 5-6 decades o f i n t e n s i t y so t h a t i n d i v i d u a l spot i d e n t i t y a n d p o s i t i o n at lower intensities c o u l d be c o r r e c t l y identified at the h i g h e r i n t e n s i t y levels w h i c h enhanced fainter spots. T h e transparencies were a l l r e p h o t o g r a p h e d w i t h a l o w c o n t r a s t film developer s y s t e m a n d p r i n t e d at the same m a g n i f i c a t i o n w i t h 0-2 grade I l f o r d m u l t i g r a d e II p r i n t p a p e r . T h e features of these d i f f r a c t i o n p a t t e r n s were recorded o n overlayed t r a c i n g paper o n a l i g h t t a b l e . T h e distance between centers of spots s y m m e t r i c w i t h the d i f f r a c t i o n p a t t e r n center were measured w i t h a vernier caliper a n d t h e n d i v i d e d b y two t o give the spot p o s i t i o n i n r e c i p r o c a l space. T h e exact center of most elongated spots was e s t i m a t e d . T h e p r e c i s i o n o f the d i f f r a c t i o n measurements was n o t better t h a n 8%. T h e r e c i p r o c a l space d i s t a n c e for the molecule was d i v i d e d i n t o the r e c i p r o c a l distance for the 1 m m g r a p h paper a n d m u l t i p l i e d b y a m a g n i f i c a t i o n factor i n À / m m t o c o m p u t e o p t i c a l d i f f r a c t i o n spacings i n A n g s t r o m s . Results I m a g i n g A. xylinum's cellulose r i b b o n has p r e v i o u s l y been a c c o m p l i s h e d b y a d h e r i n g cells t o a film coated g r i d , g r o w i n g the cells o n a n u t r i e n t buffer s o l u t i o n , a n d negative s t a i n i n g t h e m for v i s i b i l i t y a n d for t r a n s m i s s i o n electron microscopy. Images show t h a t A. xylinum produces a lefth a n d t w i s t e d r i b b o n n o r m a l l y , a n d i n the presence o f c a r b o x y m e t h y l c e l l u lose ( C M C ) ( 4 , 5 ) . W h e n g r o w n i n the presence of 0.25 m M C a l c o f l u o r , t h i s m o r p h o l o g y is d r a m a t i c a l l y altered to a b r o a d cellulose b a n d c o m p o s e d of 15Â a n d larger filaments (2-5). W e have a p p r o a c h e d s p e c i m e n p r e p a r a t i o n for T E M i m a g i n g differently. A. xylinum n a t u r a l l y forms a pellicle or gel of cellulose r i b b o n s o n the surface o f a s o l u t i o n d u r i n g g r o w t h w i t h the cells t e t h e r e d at the p e l l i c l e ' s p e r i p h e r y b y r i b b o n s . Since sequential g r o w t h c o n d i t i o n s are recorded l i n e a r l y a l o n g a r i b b o n , A. xylinum was g r o w n n o r m a l l y , i n 0.25 m M T i n o p a l , a n d n o r m a l l y a g a i n , t h e n v i s u a l i z e d after f r e e z e - d r y i n g a n d P t - C r e p l i c a t i o n . F i g u r e 1 shows a n o r m a l l y g r o w n pellicle o f A. xylinum cellulose r i b b o n s t h a t a p p e a r l i n e a r a n d u n t w i s t e d . T h e s m a l l a r r o w s p o i n t o u t lefth a n d t w i s t e d m i c r o f i b r i l s w i t h i n the r i b b o n s . W h e n A. xylinum was g r o w n i n the presence o f 0.25 m M T i n o p a l , a n altered cellulose was p r o d u c e d as s h o w n i n F i g u r e 2. T h e same p r e p a r a t i o n s h o w n at h i g h e r m a g n i f i c a t i o n i n F i g u r e 3 revealed 33Â P t - C coated s u b m i c r o f i b r i l s ( s m a l l a r r o w s ) . T h e s e i n d i v i d u a l s u b m i c r o f i b r i l s averaged 1 7 . 8 ± 2 . 2 Â after c o r r e c t i o n for the P t - C coat (1), a n d f r e q u e n t l y t w i s t e d together to f o r m larger fibrils. S u b m i c r o f i b r i l s , p r e v i o u s l y i m a g e d , have been c o r r e l a t e d w i t h the four g l u c a n c h a i n X - r a y fiber d i f f r a c t i o n u n i t cell as o p p o s e d to the two or eight c h a i n u n i t cell b y average d i a m e t e r measurements (1). I n the F i g u r e 4 s u b m i c r o f i b r i l m o d e l the side a n d d i a g o n a l d i m e n s i o n s of the u n i t cell were e s t i m a t e d . B y t r a n s l a t i n g a n d r o t a t i n g t h i s p a r a l l e l n i n e g l u c a n c h a i n cross s e c t i o n , the l o n g d i a g o n a l o f 23Â a n d the short d i a g o n a l of 19Â generated a m a j o r a n d m i n o r surface ridge a n d also generate a s u b m i c r o f i b r i l w i t h a



F i g u r e 1. F r e e z e - d r i e d gel of A. xylinum cellulose r i b b o n s d e p o s i t e d d u r i n g n o r m a l g r o w t h . T h e arrows p o i n t to t r i p l e - s t r a n d e d l e f t - h a n d h e l i c a l m i crofibrils averaging 36.8 ± 3 A i n d i a m e t e r (1). T h e s a m p l e was r e p l i c a t e d w i t h 17.3Â P t - C a n d backed w i t h 90.2Â of c a r b o n .



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F i g u r e 2. Freeze-dried A. xylinum cellulose g r o w n i n the presence of 0.25 m M T i n o p a l a n d r e p l i c a t e d w i t h 16.4Â P t - C a n d backed w i t h 156Â of c a r b o n . A t a n g l e d mass o f 33Â P t - C coated s u b m i c r o f i b r i l s was f o r m e d i n s t e a d o f n o r m a l r i b b o n cellulose.



F i g u r e 3. H i g h e r m a g n i f i c a t i o n of A. xylinum cellulose g r o w n i n the presence of 0.25 m M T i n o p a l . T h e arrows p o i n t to single P t - C coated s u b m i c r o f i b r i l s averaging 33Â i n d i a m e t e r (17.8 ± 2.2Â after c o r r e c t i o n for the 16.4Â P t C c o a t i n g ) . M a n y of these s u b m i c r o f i b r i l s were t w i s t e d together f o r m i n g thicker fibers.



F i g u r e 4. T h e m o d e l c o n t a i n s the four c h a i n X - r a y fiber d i f f r a c t i o n u n i t cell d i m e n s i o n s , l i s t e d at the left of the figure, w h i c h were d r a w n as a cross section t h r o u g h the g l u c a n chains. T h i s u n i t cell has encompassed n i n e g l u c a n chains w i t h exterior side d i m e n s i o n s of 15Â a n d 17Â a n d d i a g o n a l d i m e n s i o n s of 19Â a n d 23Â as s h o w n . B y r o t a t i n g a n d t r a n s l a t i n g the u n i t cell we generated a s u b m i c r o f i b r i l ( S M ) geometry w i t h a m a j o r ridge ( 2 3 Â diagonal) a n d a n i n t e r m e d i a t e m i n o r ridge (19Â d i a g o n a l ) . M a j o r ridges o c c u r every 36Â w i t h a h e l i c a l p i t c h of 72Â. T h e o r i g i n a l s u b m i c r o f i b r i l average measurement f r o m m i c r o g r a p h s was 1 7 . 8 ± 2 . 2 Â , i n agreement w i t h the 18.5Â f o u n d b y averaging the exterior sides a n d d i a g o n a l s . T h e average of these d i m e n s i o n s was s l i g h t l y larger t h a n o r i g i n a l l y r e p o r t e d (1) since the m i n o r d i a g o n a l was increased f r o m 16.5Â to 19Â. G e n e r a t i o n of a n ordered s u b m i c r o f i b r i l b y t h i s a p p r o a c h was consistent w i t h the E l l i s a n d W a r w i c k e r u n i t cell d e r i v a t i o n w h i c h o n l y assumed p a r a l l e l g l u c a n c h a i n axes (6).



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n o n u n i f o r m d i a m e t e r whose m a x i m u m d i m e n s i o n is 23Â. T h i s s u b m i c r o f i b r i l h a d a h e l i c a l p i t c h of 72Â w i t h a m a j o r ridge crossing the axis every 36Â a n d a m i n o r ridge crossing h a l f - w a y between the m a j o r ridges. I n F i g u r e 5 the s u b m i c r o f i b r i l m o d e l ( F i g . 5a) was c o m p a r e d at equivalent m a g n i f i c a t i o n t o a T E M i m a g e of the s u b m i c r o f i b r i l ( F i g . 5c). T h e P t - C coated 36Â m a j o r ridge spacings a n d some of the m i n o r ridges a l o n g the s u b m i c r o f i b r i l surface were preserved b u t the d i a m e t e r increased b y 15.2Â to 33Â. T h e i m a g e o r i e n t a t i o n w i t h the c o m p u t e r generated o p t i c a l d i f f r a c t i o n p a t t e r n s i n F i g u r e s 5-7 were also preserved. T h e m o d e l ' s d i f f r a c t i o n p a t t e r n ( F i g . 5b) was generated f r o m one side of a left-handed h e l i x w i t h p r o m i n e n t spacings ( ± 8 % precision) at 25Â a n d 12Â, w i t h a v e r t i c a l s p a c i n g at 12Â a n d w i t h adjacent s p o t s p a r a l l e l i n g the 25Â a n d 12Â s p o t p o s i t i o n s . T h e o p t i c a l diffraction p a t t e r n generated f r o m the T E M image of the s u b m i c r o f i b r i l ( F i g . 5d) was s i m i l a r to F i g . 5b w i t h left-handed spacings (±8%*) of 26Â a n d 12Â or 14Â a n d w i t h two spots below a n d above the larger s p a c i n g of 26À ( see h o r i z o n t a l arrows i n F i g . 5d a n d 5b). T h e m a j o r ridges spaced 26À a p a r t traverse the s u b m i c r o f i b r i l axis at a 45 ± 15° angle. T h i s angle has g e o m e t r i c a l l y d e t e r m i n e d the m a j o r ridge repeat a l o n g the fiber a x i s of 36Â (26Â / s i n 45° = 3 6 . 8 À ) . T h e P t - C r e p l i c a r e s o l u t i o n i n F i g u r e 5 d is 9 À was o n l y s l i g h t l y better t h a n the 12Â or 13Â p r e v i o u s l y r e p o r t e d ( 1 0 , 1 3 ) . T h e m i c r o f i b r i l m o d e l c o m p o s e d of three s u b m i c r o f i b r i l s l e f t - h a n d t w i s t e d together i n F i g u r e 6 a was c o m p a r e d at the same m a g n i f i c a t i o n to the P t - C coated m i c r o f i b r i l image i n F i g u r e 6c. T h e m a j o r a n d m i n o r ridges ( t h i c k a n d t h i n a r r o w heads) were v i s i b l e a l o n g the s u b m i c r o f i b r i l s i n the m o d e l a n d they c o u l d also be seen a l o n g the c e n t r a l s u b m i c r o f i b r i l i n the T E M i m a g e . T h e c o m p u t e r generated o p t i c a l diffraction p a t t e r n s of the m o d e l s h o w n i n F i g u r e 6b a n d of the P t - C coated m i c r o f i b r i l s h o w n i n F i g u r e 6d were c o m p l e x b u t s i m i l a r . In the diffraction p a t t e r n i n F i g u r e 6b, we c o u l d assign the v e r t i c a l s p a c i n g i n the m i c r o f i b r i l m o d e l at 23Â a n d one l e f t - h a n d e d s p a c i n g at 23Â (—45° ± 1 5 ° angle) to the l o n g d i a g o n a l (see F i g . 4) or m a x i m u m d i a m e t e r of the s u b m i c r o f i b r i l . T h e t h i r d 23Â s p a c i n g at - 1 7 ° ± 15° angle was p r o b a b l y related t o a foreshortened p r o j e c t i o n of the m a j o r ridge center to center distance a l o n g the s u b m i c r o f i b r i l w r a p p e d a r o u n d the m i c r o f i b r i l a x i s . T h e v e r t i c a l s p a c i n g i n F i g u r e 6 d of the T E M i m a g e was 27Â a n d there was also a left-handed s p a c i n g at 27Â at a —45° ± 15° angle. T h i s larger value m a y be due to the s l i g h t l y greater s e p a r a t i o n between the s u b m i c r o f i b r i l s i n the T E M i m a g e . A t h i r d s p a c i n g at 23Â or 21Â at a —17° ± 1 5 ° angle was p r o b a b l y related to a foreshortened m a j o r ridge s p a c i n g a l o n g the s u b m i c r o f i b r i l . T h e negatively s t a i n e d m i c r o f i b r i l s h o w n i n F i g u r e 7 a was first treated to remove hemicellulose before i t was negatively s t a i n e d . T h e c o m p u t e r generated o p t i c a l diffraction p a t t e r n was o n l y of the u p p e r m i c r o f i b r i l w i t h the 33À s p a c i n g . T h e diffraction p a t t e r n i n F i g u r e 7b showed a v e r t i c a l s p a c i n g at 24À, a left-handed s p a c i n g of 23Â at a —45° ± 5° angle t o the h o r i z o n t a l a x i s , a n d a s p a c i n g at 25Â s i m i l a r to the m a j o r ridge s p a c i n g i n F i g u r e 6b. T h e 33Â left-handed a x i a l m i c r o f i b r i l s p a c i n g was g e o m e t r i c a l l y related to the 23Â s u b m i c r o f i b r i l s p a c i n g (23Â / s i n 45° = 3 2 . 5 Â ) .



Figure 5. The submicrofibril model in a and the Pt-C coated submicrofibril image in c were compared at the same magnification. The optical diffraction of the model in b demonstrated a left-handed surface spacing of 25A and 12Â with two spots (horizontal arrows) which appeared on layer lines below and above the 25Â spot. The first layer line occurs at 12Â and was the vertical separation of the minorridgeposition between two majorridges23Â apart. The general features of this lefthanded optical diffraction pattern occurred in the TEM image in d with spacings at 26Â and 12A or 14Â with similar spots indicated by horizontal arrows below and above the major ridge spacing of 26A. (c reproduced with permission from réf. 1. Copyright 1987 Elsevier.)



Figure 6. This model of the microfibril (M) composed of three submicrofibrils (SM) twisted together in a left-handed fashion (a) was compared at the same magnification to the Pt-C coated microfibril image in c. The major and minor ridges (thick and thin arrowheads) were visible along the submicrofibrils in the model and they could also be seen along a submicrofibril at the center of the TEM image. The optical diffraction pattern of the model shown in b and of the Pt-C coated microfibril shown in d were complex but similar. In d, the submicrofibrils cross the TEM microfibril axis at a 45β ± 15 ° angle, (c reproduced with permission from réf. 1. Copyright 1987 Elsevier.)



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F i g u r e 7. T h i s m i c r o f i b r i l was treated w i t h hot trifluoroacetic a c i d to remove hemicellulose a n d was t h e n negatively s t a i n e d w i t h 2 % u r a n y l acetate i n F i g u r e 7 a . T h e o p t i c a l diffraction p a t t e r n i n F i g u r e 7b was o n l y of the u p p e r m i c r o f i b r i l s h o w i n g the 33Â s p a c i n g . In F i g u r e 7b the s u b m i c r o f i b r i l s cross the T E M m i c r o f i b r i l axis at a 45° ± 5 ° angle. (7a r e p r o d u c e d w i t h p e r m i s s i o n f r o m Réf. 1. © 1987 E l s e v i e r Science P u b l i s h e r s Β. V . )



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It was p r e v i o u s l y p o i n t e d out t h a t filaments the size of s u b m i c r o f i b r i l s exit the cell w a l l of A. xylinum t h r o u g h pores (4). F i g u r e 8 shows how a m i c r o f i b r i l self-assembled f r o m three s u b m i c r o f i b r i l s o n the e x t e r i o r of the cell (1). A r r o w s p o i n t to s u b m i c r o f i b r i l s 1 a n d 2 o n the cell surface. A t their j u n c t i o n s u b m i c r o f i b r i l 2 crosses 1 i n a left-handed m a n n e r v i s i b l e i n stereo-micrographs (not s h o w n ) . S u b m i c r o f i b r i l 3 j o i n s a n d t h e n crosses the twisted p a i r of m i c r o f i b r i l s i n a left-handed m a n n e r near the b o t t o m of the figure. T h e image of s u b m i c r o f i b r i l 3 also showed t h a t i t was not r o d - l i k e b u t was l e f t - h a n d s u p e r - t w i s t e d . A m o d e l of t h i s process i n F i g ure 9 showed three l e f t - h a n d h e l i c a l s u b m i c r o f i b r i l s l a b e l l e d S M 1, 2 a n d 3 emerging f r o m the cell w a l l at the top of the figure. T h i s m o d e l depicts three associated s u b m i c r o f i b r i l s being s p u n together to f o r m a m i c r o f i b r i l , a l t h o u g h i t was unclear how three s u b m i c r o f i b r i l s i n i t i a l l y came together. T h i s s p i n n i n g process m a y be d r i v e n b y the l e f t - h a n d r o t a t i o n a n d e l o n g a t i o n of the s u b m i c r o f i b r i l s d u r i n g cellulose synthesis. T h e left r o t a t i o n of each s u b m i c r o f i b r i l drives the f o r m a t i o n of a l e f t - h a n d three-stranded m i c r o f i b r i l w h i c h also left r o t a t e d as i t grows longer. If any of the s u b m i c r o f i b r i l s elongated more r a p i d l y t h a n the other p a i r , i t w o u l d become l e f t - h a n d s u p e r - t w i s t e d as p o i n t e d out i n F i g u r e 8 for s u b m i c r o f i b r i l 3. In t o b a c c o p r i m a r y cell w a l l the cellulose m i c r o f i b r i l s observed i n d i v i d u a l l y or associated w i t h bundles were also t r i p l e - s t r a n d e d a n d l e f t - h a n d h e l i c a l . These observations are s h o w n i n F i g u r e 10. Since cellulose is o n l y 1 9 % of the tobacco cell w a l l (17), the task of finding a n d i d e n t i f y i n g cellulose was c o m p l i c a t e d . F o r t h i s reason A. xylinum w h i c h p r o d u c e s a pure r i b b o n of cellulose was used for s t u d y i n g cellulose s t r u c t u r e . Discussion and

Conclusions

S u b m i c r o f i b r i l a n d t r i p l e - s t r a n d e d l e f t - h a n d h e l i c a l m i c r o f i b r i l s are f o u n d i n t o b a c c o p r i m a r y cell w a l l a n d b a c t e r i a l A. xylinum cellulose. W e suspect f r o m our results a n d the l i t e r a t u r e survey o u t l i n e d i n reference (1) t h a t the t r i p l e s t r a n d e d structures are p r o m i n e n t i n the p r i m a r y p l a n t cell w a l l . T h e h i g h l y c r y s t a l l i n e cellulose of p l a n t a n d algae secondary cell w a l l appears b y X - r a y fiber diffraction (18,19) a n d T E M l a t t i c e i m a g i n g (20-23) to be largely c r y s t a l l i n e a r r a y s of p l a n a r s t r a i g h t chains of ( l - 4 ) - / ? - D - g l u c a n chains. T h e s u b m i c r o f i b r i l i n F i g u r e 5c was clearly a left-handed h e l i x w i t h a m a j o r ridge repeat of 26Â a n d m i n o r ridge h a l f s p a c i n g at 12Â or 14Â evident i n the o p t i c a l diffraction p a t t e r n ( F i g . 5d). T h e correspondence between the m o d e l a n d the P t - C coated s u b m i c r o f i b r i l was stronger t h a n expected since i t was k n o w n t h a t evaporated m e t a l coatings do not j u s t adhere where they l a n d at o r d i n a r y r e p l i c a t i o n temperatures (24-27). T h e specimen t e m p e r a t u r e used i n t h i s w o r k (—178°C) was colder b y 110°C t h a n the t e m p e r a t u r e used p r e v i o u s l y (7). T h e greater m e t a l s t i c k i n g coefficient at lower temperatures preserved surface resolution to 9Â i n F i g u r e 5 d . T h e m i c r o f i b r i l m o d e l i n F i g u r e 6a s t r o n g l y resembled the P t - C coated m i c r o f i b r i l i n F i g u r e 6c, w h i c h also c o n t a i n e d a m a j o r a n d m i n o r ridge a l o n g a s u b m i c r o f i b r i l at the center of the image. T h e assignment of s p o t s to molecular features i n the c o m p l e x o p t i c a l diffraction p a t t e r n i n F i g u r e 6b



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F i g u r e 8. Since the s u b m i c r o f i b r i l s exit the cell w a l l o f A. xylinum t h r o u g h pores (4), the self-assembly of a t r i p l e - s t r a n d e d m i c r o f i b r i l has o c c u r r e d at the e x t e r i o r surface of the cell (1). S u b m i c r o f i b r i l s 1 a n d 2 a p p e a r e d s u p e r t w i s t e d o n the cell surface. A t t h e i r j u n c t i o n s u b m i c r o f i b r i l 2 crossed 1 i n a l e f t - h a n d e d m a n n e r w h i c h is o n l y v i s i b l e w i t h s t e r e o - m i c r o g r a p h s (not s h o w n ) . S u b m i c r o f i b r i l 3, w h i c h was also l e f t - h a n d s u p e r - t w i s t e d , j o i n e d a n d crossed the double fiber i n a l e f t - h a n d e d m a n n e r . T h i s s p e c i m e n was coated w i t h 16.4Â o f P t - C . ( R e p r o d u c e d w i t h p e r m i s s i o n f r o m Réf. 1. © E l s e v i e r Science P u b l i s h e r s Β. V . )




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F i g u r e 9. T h i s m o d e l shows three left-handed h e l i c a l s u b m i c r o f i b r i l s ( S M ) 1, 2 a n d 3 w h i c h emerged f r o m the cell w a l l at their t e r m i n i . It was not clear how the s u b m i c r o f i b r i l s first associated w i t h other s u b m i c r o f i b r i l s b u t once associated they were s p u n together. T h i s m o d e l assumed t h a t cellulose synthesis p r o v i d e d the m e c h a n i c a l force t h a t s i m u l t a n e o u s l y extended a n d l e f t - h a n d r o t a t e d the s u b m i c r o f i b r i l s , w h i c h i n t u r n drove the secondary f o r m a t i o n of three s u b m i c r o f i b r i l s into a l e f t - h a n d h e l i c a l m i c r o f i b r i l ( M ) .



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c o u l d not be a c c o m p l i s h e d yet except at a r u d i m e n t a r y l e v e l . T h e v e r t i c a l a n d l e f t - h a n d e d s p a c i n g (—45° ± 15°) at 23Â represented the m a x i m u m w i d t h or l o n g d i a g o n a l ( F i g . 4) of the s u b m i c r o f i b r i l . T h e —17° ± 15° spaci n g at 23Â p r o b a b l y represented a foreshortened m a j o r ridge s p a c i n g a l o n g a s u b m i c r o f i b r i l c u r v i n g a r o u n d the m i c r o f i b r i l a x i s . T h e same spacings also a p p e a r e d i n the o p t i c a l diffraction p a t t e r n ( F i g . 7b) of the n e g a t i v e l y s t a i n e d m i c r o f i b r i l i n F i g u r e 7 a . T h e o p t i c a l d i f f r a c t i o n p a t t e r n o f the freeze-dried P t - C r e p l i c a t e d m i c r o f i b r i l ( F i g . 6d) was far m o r e c o m p l e x , res o l v i n g m o r e s p o t s t h a n its negatively s t a i n e d c o u n t e r p a r t i n F i g u r e 7b. T h e v e r t i c a l a n d l e f t - h a n d e d spacings at —45° ± 1 5 ° i n the P t - C coated m i c r o f i b r i l were 27Â, due either t o a t r u l y greater space between s u b m i crofibrils i n F i g u r e 6c or m e r e l y to the 8% p r e c i s i o n o f measurement w h i c h m a d e 27À a n d 23Â of d o u b t f u l d i s c r i m i n a b i l i t y . A 21À or 23Â s p a c i n g was also l o c a t e d at —17° ± 15°, w h i c h c o u l d be a t t r i b u t e d t o the s u b m i c r o f i b r i l . W e believe t h a t the T E M a n d m o d e l images of the cellulose h e l i x i n c o n j u n c t i o n w i t h the c o m p u t e r generated o p t i c a l d i f f r a c t i o n p a t t e r n s p r o v i d e s t r o n g evidence for a s u b s t r u c t u r e t h a t includes a l e f t - h a n d h e l i c a l m i c r o f i b r i l c o m p o s e d of three s u b m i c r o f i b r i l s ( F i g . 8) a n d a l e f t - h a n d h e l i c a l s u b m i c r o f i b r i l . E v i d e n c e t h a t the four g l u c a n c h a i n fiber d i f f r a c t i o n u n i t cell h a d the same size as the s u b m i c r o f i b r i l came f r o m the correspondence between the u n i t cell's average diameter of 18.5Â a n d the measured s u b m i c r o f i b r i l d i a m e t e r of 17.8 ± 2.2Â (1), the m a j o r a n d m i n o r ridges v i s i b l e i n T E M ( F i g . 5c) a n d the m a x i m u m d i a m e t e r of 23Â for the s u b m i c r o f i b r i l as d e t e r m i n e d f r o m the m i c r o f i b r i l ' s o p t i c a l diffraction p a t t e r n . T h i s s t r o n g c o r r e l a t i o n has s u p p o r t e d the hypothesis t h a t the p a r a l l e l n i n e g l u c a n c h a i n u n i t represents the s u b m i c r o f i b r i l cross s e c t i o n . T h e m e t h o d we have advanced for g e n e r a t i n g the s u b m i c r o f i b r i l f r o m the n i n e sugar u n i t cross section i n F i g u r e 4 provides a m o d e l for s u b m i c r o f i b r i l synthesis. A f t e r each nine g l u c a n c h a i n cross section has been assembled a n d m o v e d to make r o o m for the n e x t , the s u b m i c r o f i b r i l e l o n gates a n d s i m u l t a n e o u s l y rotates. M i c r o f i b r i l self-assembly i n F i g u r e 8 was based o n a cellulose synthesis-powered m e c h a n i s m w h i c h extends a n d r o tates each s u b m i c r o f i b r i l i n the F i g u r e 9 m o d e l . O n e obvious p r e d i c t i o n of the m o d e l , besides the f o r m a t i o n of a l e f t - h a n d h e l i c a l m i c r o f i b r i l , was t h a t the m i c r o f i b r i l w o u l d also rotate i n a l e f t - h a n d e d d i r e c t i o n as i t e l o n g a t e d . W e w i l l r e t u r n to t h i s p o i n t i n the last p a r a g r a p h . O n e i m p o r t a n t consequence of the n i n e p a r a l l e l g l u c a n c h a i n u n i t , t r a n s l a t e d a n d r o t a t e d to generate a l e f t - h a n d h e l i c a l s u b m i c r o f i b r i l , was t h a t a l l the ( l - 4 ) - / ? - D - g l u c a n chains were not c o n f o r m a t i o n ally equivalent i n the F i g u r e 4 s u b m i c r o f i b r i l m o d e l . F o r instance, the g l u c a n c h a i n at the center of t h i s m o d e l w o u l d f o r m a l e f t - h a n d e d h e l i x 7 cellobiose u n i t s l o n g w i t h a 72À p i t c h , b u t a l l other g l u c a n chains w o u l d require m o r e cellobiose u n i t s to reach the same a x i a l p o s i t i o n . Recent t h e o r e t i c a l c a l c u l a t i o n s i n d i c a t e t h a t a f a m i l y of l e f t - h a n d h e l i c a l ( l - 4 ) - / ? - D - g l u c a n chains 7-9 cellobiose u n i t s l o n g w i t h pitches of a b o u t 72Â to 93Â exist near a s i m i l a r c o n f o r m a t i o n a l energy m i n i m u m (28). T h e nine p a r a l l e l g l u c a n c h a i n , t w i s t e d c r y s t a l m o d e l thus cannot be r u l e d out o n either the basis of g l u c a n



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Figure 10. a, Bundled cellulose microfibrils in the lower epidermal cell wall (facing mesophyll cells) of Coker 319 tobacco leaves. This epidermal peel was freeze-dried, Pt-C replicated (15.9 Â thick) and carbon film backed (133 Â thick), b, A cellulose microfibril is seen connecting two bundles of microfibrils (similar to a). This Pt-C coated microfibril averages 51 Â in width, shows left-handed surface striations, and splits into three smaller submicrofibrils. c, Tobacco primary cell wall Pt-C coated microfibril averaging 50 Â shows left-handed surface striations. d, Three 16-18-Â submicrofibrils in adjacent ridges wrap (see arrows) in a left-handed fashion around the microfibril axis. Bar, 100 A . (a-d reproduced with permissionfromréf. 1. Copyright 1987 Elsevier.)



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Figure 10. e, Optical diffraction pattern of c. Its important features were similar to those in the A . xylinum microfibril diffraction pattern in Figure 6d: left-handed spacings at roughly 24 ± 3 A , a vertical spacing at 26 Λ and right-handed spacings at 36 Â and 28 Â The left-handed pattern at 47 Â and 48 A , not previously seen, was probably caused by an artificial bunching of the submicrofibrils (d) when the microfibril was lifted above the cell wall surface by peeling the lower epidermal cell layer from the tobacco leaf.



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c h a i n energy considerations or the lack of left-handed g l u c a n c h a i n conform a t i o n s . O t h e r s have also suggested t h a t cellulose g l u c a n chains f o r m a left-handed h e l i x of 72Â (29) a n d can also be left-handed h e l i c a l i n s o l u t i o n (30). T h e nonequivalence o f g l u c a n chains i n a t w i s t e d c r y s t a l w o u l d l i m i t its m a x i m u m l a t e r a l dimensions. Since chains farthest f r o m the axis center are less t w i s t e d , it is not s u r p r i s i n g t h a t i n larger secondary cell w a l l c r y s t a l l i n e cellulose a l l the g l u c a n chains can energetically assume a flat l i n e a r c o n f i g u r a t i o n w i t h each cellobiose u n i t related to the next b y a 180° r o t a t i o n (18-23,31). O u r observations suggest t h a t the s u b m i c r o f i b r i l s t r u c t u r e is a consequence of its s m a l l size a n d of ( l - 4 ) - / ? - D - g l u c a n c h a i n s ' n a t u r a l tendency t o assume a left-handed h e l i x (21). L a r g e r cellulose c r y s tals c a n u n t w i s t ( l - 4 ) - / ? - D - g l u c a n chains because of the favorable energetics of f o r m i n g p l a n a r s t r a i g h t c h a i n crystals (31). T h e m o d e l i n F i g u r e 9 predicts t h a t each m i c r o f i b r i l w o u l d rotate i n the process of cellulose r i b b o n f o r m a t i o n . If the A. xylinum cell were h e l d s t a t i o n a r y , then the r i b b o n w o u l d be l e f t - h a n d t w i s t e d (2-5); however, i f the r i b b o n were h e l d s t a t i o n a r y , then the cell w o u l d rotate (32). T h e l a t t e r case e x p l a i n s w h y r i b b o n s appear u n t w i s t e d i n the pellicle of r i b b o n s s h o w n i n F i g u r e 1. M o r e o v e r , i t has been d e m o n s t r a t e d t h a t a n A. xylinum cell ceased r o t a t i o n w h e n C a l c o f l u o r ( > 0.1 m M ) was added to the s o l u t i o n (32). P r e v i o u s w o r k has s h o w n t h a t the presence of C a l c o f l u o r or T i n o p a l c o u l d d r a m a t i c a l l y increase A. xylinum cellulose synthesis. T h i s observat i o n was the basis for the hypothesis t h a t cellulose p o l y m e r i z a t i o n can be u n c o u p l e d f r o m a slower sequential c r y s t a l l i z a t i o n step (2-5). W e believe the hypothesis is not consistent w i t h our observations. A t the very least, the presence of a n ordered a n d c r y s t a l - l i k e s u b m i c r o f i b r i l p r o d u c e d i n the presence of 0.25 m M T i n o p a l w o u l d relegate T i n o p a l ' s or C a l c o f l u o r ' s effects to a n event o c c u r r i n g after the i n i t i a l cellulose p o l y m e r i z a t i o n - c r y s t a l l i z a t i o n step or steps. T h e d a t a we present show t h a t A. xylinum cellulose m i c r o f i b r i l s are f o r m e d b y s u b m i c r o f i b r i l s b e i n g s p u n together, as s h o w n i n F i g u r e s 8 a n d 9, r a t h e r t h a n associated t h r o u g h a m e c h a n i s m of l a t e r a l f a s c i a t i o n ( 1 , 4 , 5 ) . W e have d e m o n s t r a t e d t h a t T i n o p a l d i s r u p t s r i b b o n f o r m a t i o n a n d the m i c r o f i b r i l f o r m a t i o n process i n F i g u r e s 2 a n d 3. Since the m i c r o f i b r i l s p i n n i n g process either rotates the r i b b o n or the c e l l , its d i s r u p t i o n w o u l d u n c o u p l e a r a t e - l i m i t i n g r o t a t i o n , e l o n g a t i o n process f r o m cellulose synthesis. T h u s , u n c o u p l i n g of a cell's or a r i b b o n ' s m e c h a n i c a l r o t a t i o n w i t h T i n o p a l or C a l c o f l u o r w o u l d result i n a n increased rate of cellulose synthesis, a n i n t e r p r e t a t i o n w h i c h is consistent b o t h w i t h our findings a n d w i t h previous work (1-5,32). Acknowledgments W e acknowledge P h i l i p M o r r i s ' financial support for t h i s w o r k , N . J . J a cobs ( D a r t m o u t h M e d i c a l School) for c u l t u r i n g the A. xylinum, a n d the D a r t m o u t h R i p p e l E M F a c i l i t y for the use of their e q u i p m e n t .


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