Structural Characterization and Visualization In Situ and After Isolation

Jul 23, 2009 - The presence of rhamnose in the backbone of pectin was believed to create "kinks" which probably disrupted helical stretches of the 4-l...
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Chapter 21 Structural Characterization and Visualization In Situ and After Isolation of Tobacco Pectin 1

George C. Ruben and Gordon H. Bokelman

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Department of Biology, Dartmouth College, Hanover, NH 03755 Philip Morris USA, Research Center, Richmond, VA 23234 2

Recently two different disciplines, chemical structural elucidation and transmission electron microscopy, were utilized in the study of pectin, with particular empha­ sis on tobacco pectin. The goal was to help bridge the gap between knowledge of their chemical structures to understanding the complex physical structures re­ vealed by microscopy. To provide background on chem­ ical structure, a study established that tobacco pectin was present as a series of related rhamnogalacturonans. All of these polysaccharides had a backbone consist­ ing of 4-linked α-D-galactopyranosyluronic acid residues interspersed with 2-linked L-rhamnopyranosyl residues. However, they varied in content of neutral sugars and extent of methyl-esterification. The presence of rham­ nose in the backbone of pectin was believed to create "kinks" which probably disrupted helical stretches of the 4-linked α-D-galactopyranosyluronic acid residues. In the present study pectin samples were gelled in deionized water, air-dried or freeze-dried, platinum-carbon repli­ cated, carbon-backed and then examined by high reso­ lution transmission electron microscopy. The pectin was found to be present as single chains of 7 ± 3Å diameter that showed helical stretches with a 13Å left-handed surface striation. P e c t i n is the m a j o r c o m p o n e n t f o u n d i n the p r i m a r y cell walls o f dicots a n d m a y p l a y a v i t a l role i n cell g r o w t h . D u r i n g cell g r o w t h , loosening o f the cell w a l l b y a c i d i f i c a t i o n is a n i m p o r t a n t process w h i c h enables the cell t o elongate b y its o w n t u r g o r pressure. It has been suggested t h a t the p r i m a r y a c t i o n o f a c i d i f i c a t i o n is the loosening o f a c a l c i u m pectate gel w i t h i n t h e 0097-6156/89/0399-0300$06.00/0 © 1989 American Chemical Society

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cell w a l l (1). P e c t i n is also f o u n d i n the m i d d l e l a m e l l a of l a n d p l a n t tissues where it is t h o u g h t to f u n c t i o n as a n i n t e r c e l l u l a r b i n d i n g agent (2). P e c t i n constitutes 1 1 - 1 2 % of the t o t a l solids (3) or 3 4 % of the cell w a l l m a t e r i a l s (4) i n t o b a c c o l a m i n a . B y c o n t r a s t , a l l of the f o l l o w i n g c o m ­ ponents are f o u n d to a lesser extent w i t h i n the cell walls of t o b a c c o l a m ­ i n a : p r o t e i n ( 2 1 . 6 % ) , cellulose ( 1 8 . 7 % ) , hemicellulose (11.4%), a n d l i g n i n (4.1%). G a l a c t u r o n i c a c i d is the m a j o r c o n s t i t u e n t of a l l n a t u r a l p e c t i n s . P e c t i n s also c o n t a i n v a r y i n g quantities of n e u t r a l sugars, p r i n c i p a l l y a r a b i nose, galactose a n d rhamnose (5). T h e c a r b o x y l f u n c t i o n o f the g a l a c t u r o n o s y l residues m a y be present as a m e t h y l ester, a c i d or s a l t . P e c t i n s are best k n o w n for t h e i r a b i l i t y to f o r m gels (6), a p r o p e r t y w h i c h often involves i n t e r m o l e c u l a r b i n d i n g m e d i a t e d by c a l c i u m cations (7). T h e p r i n c i p a l c o m m e r c i a l use of p e c t i n is i n the p r e p a r a t i o n of j e l l y a n d j a m p r o d u c t s (8). P e c t i n s provide firmness i n fresh f r u i t s a n d vegetables (9-11). H i s t o r i c a l l y , n a t u r a l tobacco pectins also have been used as b i n d e r s to prepare r e c o n s t i t u t e d sheets f r o m t o b a c c o b y - p r o d u c t s t h a t are t h e n i n c o r p o r a t e d i n t o cigarette filler or cigar w r a p p e r s (12-14). P e c t i n s have been s t r u c t u r a l l y characterized b y a c o m b i n a t i o n of c h e m ­ i c a l a n d spectroscopic m e t h o d s . C N M R can be used to e x a m i n e the p u ­ r i t y , degree o f e s t e r i f i c a t i o n , a n d n e u t r a l sugar content o f p e c t i n s (15). T h e m o n o m e r i c c o m p o s i t i o n of pectins m a y be d e t e r m i n e d d i r e c t l y b y a c o m b i ­ n a t i o n of m e t h a n o l y s i s (16) a n d s i l y l a t i o n procedures to y i e l d O - s i l y l a t e d m e t h y l g l y c o s i d e s t h a t can be q u a n t i t a t e d by G C (15). T h e linkage p a t ­ tern of the m o n o m e r i c sugars m a y be d e t e r m i n e d b y m e t h y l a t i o n a n a l y s i s . T h i s procedure involves m e t h y l a t i o n of the s t a r t i n g p e c t i n b y the H a k o m o r i m e t h o d (17-19), r e d u c t i o n of the c a r b o x y l i c a c i d f u n c t i o n s (18), h y d r o l y ­ sis, r e d u c t i o n of the aldehyde functions a n d a c e t y l a t i o n to y i e l d p a r t i a l l y m e t h y l a t e d a l d i t o l acetates. T h e p a r t i a l l y m e t h y l a t e d a l d i t o l acetates t h e n can be a n a l y z e d b y G C / M S (20). I n a v a r i a t i o n of the procedures l i s t e d above, p a r t i a l a c i d h y d r o l y s i s m a y be used to generate a series of d i - a n d oligosaccharide derivatives (15). These derivatives can be i d e n t i f i e d b y t h e i r electron i m p a c t mass s p e c t r a l f r a g m e n t a t i o n (21). F r o m a l l of the above i n f o r m a t i o n the c h e m i c a l s t r u c t u r e of the s t a r t i n g p e c t i n t h e n m a y be de­ duced. 1 3

T h e results f r o m s t r u c t u r a l studies on pectins isolated f r o m a n u m b e r of different p l a n t sources have been r e p o r t e d i n several papers a n d review articles ( 1 0 , 2 2 - 2 8 ) . C h e m i c a l investigations o f t o b a c c o p e c t i n ( 1 5 , 2 9 - 3 1 ) have d e m o n s t r a t e d t h a t its s t r u c t u r e is consistent w i t h the basic s t r u c t u r a l elements f o u n d i n pectins f r o m other sources. I n one recent s t u d y (15), i s o l a t i o n a n d p u r i f i c a t i o n o f t o b a c c o p e c t i n y i e l d e d a series of related r h a m n o g a l a c t u r o n a n s . A l l of these p o l y s a c ­ charides were f o u n d to have a backbone c o n s i s t i n g of 4 - l i n k e d a - D g a l a c t o p y r a n o s y l u r o n i c a c i d residues interspersed w i t h 2 - l i n k e d L - r h a m n o p y r a n o s y l residues i n a r a t i o of ~ 16:1 (see F i g . 1). T h e presence of r h a m ­ nose i n the b a c k b o n e of p e c t i n is believed to create " k i n k s " w h i c h p r o b a b l y d i s r u p t h e l i c a l stretches of the 4 - l i n k e d α-D-galactopyranosyluronic a c i d

=

=

=

=

Rha

G al A

(5-Me-GalA

R.

2 ) - I l h a - (1 -

- GalA -

^ - -(—4)

- 6 - Me - G a l A - ( l - )

F i g u r e 1. M o d e l c h e m i c a l s t r u c t u r e of t o b a c c o p e c t i n .

ι

^

^

terminal /?-D-galactopyranosyl residue

4-linked /?-D-galactopyranosyl residue

C

(-

2j

Rha -

terminal α-L-arabinofuranosyl residue

^ — 2) -

5-linked α-L-arabinofuranosyl residue

rnethyl-esterified D-galactopyranosyluronic acid residue

D-galactopyranosyluronic acid residue

L-rhamnopyranosyl residue

(—4)

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residues (10). T h e s e t o b a c c o r h a m n o g a l a c t u r o n a n s v a r i e d i n content of n e u t r a l sugars a n d extent of m e t h y l - e s t e r i f i c a t i o n . T h e i r average degree of p o l y m e r i z a t i o n was e s t i m a t e d to be 400. X - r a y fiber d i f f r a c t i o n studies have been p e r f o r m e d o n s o d i u m a n d c a l c i u m pectate gels (32). F r o m t h i s research a w o r k i n g m o d e l for the h e l i c a l p o r t i o n s of the p e c t i n c h a i n has emerged, w h i c h is i m p o r t a n t for c o m p a r i s o n to the t r a n s m i s s i o n electron m i c r o s c o p y ( T E M ) studies. T h e fiber d i f f r a c t i o n gel models assume a n t i p a r a l l e l a - ( l —• 4) p o l y g a l a c t u r o n a t e chains w h i c h , w h e n viewed d o w n the c axis of the p e c t i c a c i d u n i t cell, average about 6.8 x 7.2Â a l o n g the a a n d b directions for a single sugar c h a i n , respectively. O n e or t w o waters of h y d r a t i o n can increase t h i s size b y 3À or 6Â ( 3 3 , 3 4 ) i n deep-etched p r e p a r a t i o n s or a n associated c a l c i u m i o n c a n increase its cross-sectional d i m e n s i o n s b y a b o u t 2Â (35). F e w p r e v i o u s a t t e m p t s have been m a d e to v i s u a l i z e p e c t i n at the m o l e c u l a r level (36). I n the present s t u d y , a P t - C r e p l i c a t i o n t e c h n i q u e a n d T E M were used to characterize the u n c u t i n i z e d surface of lower e p i d e r m a l cells (facing m e s o p h y l l cells) i n b o t h fresh, green a n d senescing C o k e r 319 t o bacco leaves. These surfaces were c o m p a r e d to freeze-dried a n d a i r - d r i e d calcium-free p e c t i n gels. T h e surface textures a n d e s t i m a t e d d i a m e t e r s of single p e c t i n chains i n these p r e p a r a t i o n s were c o m p a r e d . W i t h h i g h m a g n i f i c a t i o n i m a g i n g we were able to c o n f i r m the presence o f the p o l y g a l a c t u r o n a t e c h a i n h e l i x i n tobacco a n d c i t r u s p e c t i n s . Methods and Materials T h e t o b a c c o p e c t i n used i n t h i s s t u d y was o b t a i n e d f r o m a single grade of h e a v y or b o d i e d , field-grown, flue-cured b r i g h t t o b a c c o h a r v e s t e d at the u p p e r m i d s t a l k p o s i t i o n . C r u d e tobacco p e c t i n was o b t a i n e d b y e x t r a c t i o n w i t h hot w a t e r of the t o b a c c o l a m i n a t h a t p r e v i o u s l y h a d been t r e a t e d w i t h aqueous e t h a n o l t o remove waxes, n i c o t i n e , s i m p l e sugars a n d other low m o l e c u l a r weight c o m p o n e n t s (15). T h i s crude p r o d u c t was p u r i f i e d b y t a n g e n t i a l flow u l t r a f i l t r a t i o n , i o n exchange c h r o m a t o g r a p h y a n d gel p e r m e a t i o n c h r o m a t o g r a p h y (15). T h e p u r i f i e d t o b a c c o p e c t i n h a d a g a l a c t u r o n i c a c i d content of ~ 8 0 % , a degree of esterification of ~ 22 a n d a degree of p o l y m e r i z a t i o n of ~ 400. A separate s a m p l e of deesterified p e c t i n was o b t a i n e d b y s a p o n i f i c a t i o n (15) of the p u r i f i e d tobacco p e c t i n . G e l s were o b t a i n e d i n the f o l l o w i n g m a n n e r f r o m b o t h the p u r i f i e d , s t a r t i n g t o b a c c o p e c t i n a n d the deesterified p e c t i n o b t a i n e d f r o m i t . F i r s t the s a m p l e of p e c t i n was s o l u b i l i z e d i n deionized 100°C water (~ 1% sol u t i o n ) . T h e n the p e c t i n was gelled b y e t h a n o l v a p o r , i n t r o d u c e d s l o w l y (6 hrs) by s u r r o u n d i n g the vessel c o n t a i n i n g aqueous p e c t i n w i t h 1 0 0 % e t h a n o l i n a closed container at 20° C . T h e gel f r o m the p u r i f i e d t o b a c c o p e c t i n was f o r m e d o n 1.3 c m a s h less W h a t m a n 50 filter p a p e r discs w h i c h were frozen i n p r o p a n e at a b o u t - 1 9 0 ° C . These samples were t h e n freeze-dried (90 m i n ) at —70°C, r e p l i cated w i t h 16.9Â P t / C at - 1 7 8 ° C i n a 5 x 1 0 ~ t o r r . v a c u u m a n d backed w i t h 146Â of c a r b o n . F i n a l l y , these samples were digested w i t h 8 0 % s u l furic a c i d , rinsed w i t h deionized w a t e r , p i c k e d u p f r o m u n d e r n e a t h w i t h 8

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c a r b o n - c o a t e d 300 mesh grids (37) a n d e x a m i n e d b y t r a n s m i s s i o n electron microscopy. Unless specifically noted otherwise, the same general p r o c e dures were e m p l o y e d to prepare other samples for T E M e x a m i n a t i o n . A m o r e d e t a i l e d discussion of the T E M procedures used, i n c l u d i n g m i c r o g r a p h reversals, has been p u b l i s h e d p r e v i o u s l y (37). Citrus pectin ( "Polygalacturonic A c i d M e t h y l Ester from Citrus Fruits, G r a d e I") was o b t a i n e d f r o m the S i g m a C h e m i c a l C o m p a n y . It h a d a g a l a c t u r o n i c a c i d content of ~ 8 9 % a n d a degree of esterification of ~ 57. Separate aqueous s o l u t i o n s of c i t r u s p e c t i n were freeze-dried a n d a i r - d r i e d i n deionized w a t e r . These samples were r e p l i c a t e d w i t h 9.8Â P t / C a n d backed w i t h 148Â of c a r b o n . T h e replicas for these samples were p i c k e d u p w i t h o u t a c a r b o n s u p p o r t film (38). T h e f o l l o w i n g procedure was used to o b t a i n images of the e p i d e r m a l cell surfaces w h i c h face the m e s o p h y l l cells w i t h i n the leaf i n t e r i o r . B o t h fresh green a n d senescing greenhouse-grown C o k e r 319 t o b a c c o leaves were e x a m i n e d . T h e lower e p i d e r m a l layer was peeled f r o m the underside of each leaf, rinsed t w i c e i n a s o l u t i o n of 1 : 3 / e t h a n o l : w a t e r , a n d frozen o n a | - i n . m i c a disc. T h e senescing s a m p l e was freeze-dried at —80°C for 105 m i n , r e p l i c a t e d w i t h 26.6Â P t / C a n d backed w i t h 215Â o f c a r b o n . T h e fresh, green s a m p l e was freeze-dried at — 7 0 ° C for 3 h r , r e p l i c a t e d w i t h 15.9Â P t / C a n d backed w i t h 139Â of c a r b o n . In a separate e x p e r i m e n t a s a m p l e of the lower e p i d e r m a l layer f r o m a fresh, green C o k e r 319 tobacco leaf was t r e a t e d w i t h b o i l i n g water for 25 m i n . T h i s s a m p l e was t h e n r i n s e d , freeze-dried for 3.5 h r at —80°C, r e p l i c a t e d w i t h 15.9Â P t / C a n d backed w i t h 133Â of c a r b o n . Results and Discussion Since i t was k n o w n t h a t p e c t i n can be s o l u b i l i z e d w i t h hot w a t e r , a s i m p l e e x p e r i m e n t was p e r f o r m e d to help identify the l o c a t i o n o f p e c t i n o n the n o n c u t i n i z e d surface of tobacco lower e p i d e r m a l cells. T h e n o n c u t i n i z e d surface of the lower e p i d e r m a l cells is the side w h i c h faces the m e s o p h y l l cells. F i g u r e s 2 A a n d 2 B , respectively, show the c o n t r o l a n d treated samples for the n o n c u t i n i z e d lower e p i d e r m a l cell surface of a fresh, green C o k e r 319 tobacco leaf. T r e a t m e n t consisted of i m m e r s i n g the lower e p i d e r m a l peel i n b o i l i n g water for 25 m i n . It m a y be seen i n F i g u r e 2 A t h a t the surface p e c t i n coat was continuous, w i t h n u m e r o u s flat regions. T h i s p e c t i n coat d i s a p p e a r e d f o l l o w i n g hot water e x t r a c t i o n . F i g u r e 2 B shows the exterior surface of the lower e p i d e r m i s for the b o i l i n g - w a t e r t r e a t e d s a m p l e f r o m w h i c h most of the p e c t i n h a d been e x t r a c t e d . Some p e c t i n - l i k e m a t e r i a l r e m a i n e d as s m o o t h globs c o a t i n g the filamentous p r i m a r y cell w a l l c e l l u lose. T h e contrast between F i g u r e s 2 A a n d 2 B i n d i c a t e d t h a t a p e c t i n gel permeates the cell w a l l . F i g u r e s 3 A a n d 3 B show l o w a n d h i g h m a g n i f i c a t i o n images of the n o n c u t i n i z e d lower e p i d e r m a l cell surfaces i n a senescent C o k e r 319 t o bacco leaf. T h e j u n c t i o n s between four different e p i d e r m a l cells c a n be seen i n F i g u r e 3 A . A l s o , m u l t i p l e layers o f p e c t i n were evident o n these lower e p i d e r m a l cells, b u t were not present o n the e p i d e r m a l surface of

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F i g u r e 2 A . F r e e z e - d r i e d , P t / C r e p l i c a t e d , u n t r e a t e d n o n c u t i n i z e d lower e p i d e r m a l cell surface of a f r e s h , green C o k e r 319 tobacco leaf. ( B a r = 1,000Â.)

F i g u r e 2 B . Freeze-dried, P t / C r e p l i c a t e d , n o n c u t i n i z e d lower e p i d e r m a l cell surface of a fresh, green C o k e r 319 tobacco leaf treated w i t h b o i l i n g water for 25 m i n u t e s . ( B a r = 5 , 0 0 0 A . )

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F i g u r e 3 A . Freeze-dried, P t / C r e p l i c a t e d , n o n c u t i n i z e d lower e p i d e r m a l cell surfaces i n senescent C o k e r 319 tobacco leaf. ( B a r = 5 , 0 0 0 Â . )

F i g u r e 3 B . S a m e as F i g u r e 3 A , except higher m a g n i f i c a t i o n . ( B a r = 500Â.)

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younger leaves (see F i g u r e 2 A ) . A s m a n y as 6 t o 7 stacked surface layers were seen f r o m w h i c h filaments p r o t r u d e d . T h e h i g h l y bent filaments w i t h some l i n e a r stretches were even m o r e evident i n F i g u r e 3 B . T h e average filament w i d t h was measured at 29.7Â (n = 137, S . D . = 4 . 8 Â ) . C o r r e c t i o n for the P t / C film thickness (39) gave a real size of 4 . 6 ± 4 . 8 À . F i g u r e 4 A shows a r e l a t i v e l y low m a g n i f i c a t i o n m i c r o g r a p h of a gel p r e p a r e d f r o m deesterified t o b a c c o p e c t i n . U n l i k e F i g u r e s 2 A a n d 3 A , i t s surface was smoother a n d d i d not show the l a y e r i n g effect seen o n the lower e p i d e r m a l cell surface. F i g u r e 4 B is a h i g h m a g n i f i c a t i o n m i c r o g r a p h of a gel of p e c t i n filam e n t s p r e p a r e d f r o m p u r i f i e d t o b a c c o p e c t i n . T h e average filament w i d t h w i t h P t / C c o a t i n g was 22.5Â. A f t e r c o r r e c t i n g for the a d d e d size due t o the P t / C c o a t i n g (39), the p e c t i n filament h a d a d i a m e t e r o f 7.1 ± 3Â (n = 112, S . D . = 3 Â ) . W i t h i n the s t a n d a r d d e v i a t i o n o f the m e a s u r e m e n t s , the filament w i d t h s were the same for b o t h the gel p r e p a r e d f r o m p u r i f i e d t o b a c c o p e c t i n a n d the t o b a c c o e p i d e r m a l cell surface. B o t h of these m e a surements also agreed very w e l l w i t h the x - r a y fiber d i f f r a c t i o n d i a m e t e r , ~ 7À, w h i c h we e s t i m a t e d f r o m the modeled gels of W a l k i n s h a w a n d A r n o t t (32). O n careful i n s p e c t i o n , some of the p e c t i n molecules i n F i g u r e 4 B showed a l e f t - h a n d e d surface s t r i a t i o n o c c u r r i n g every 13Â. W a l k i n s h a w a n d A r n o t t d e m o n s t r a t e d t h a t (citrus) p e c t i n c o n t a i n e d a 13.3Â 3-fold hel i x i n the p o l y g a l a c t u r o n a t e c h a i n (32), b u t x - r a y fiber d i f f r a c t i o n d i d not give the h e l i x handedness. W e report here for the first t i m e t h a t i t is a left-handed helix. Since the x - r a y fiber diffraction measurements based o n c i t r u s p e c t i n (32) were consistent w i t h the T E M measurements of t o b a c c o p e c t i n , we p r e p a r e d a gel f r o m c i t r u s p e c t i n s i m i l a r to the previous x - r a y s a m p l e . T h i s gel was t h e n e x a m i n e d b y T E M . A i r - d r i e d samples of t h i s gel, s h o w n i n F i g u r e 5, d e m o n s t r a t e d l o n g stretches of h e l i x i n the molecules l y i n g o n the surface. (In the freeze-dried g e l s — n o t s h o w n — o n l y short stretches of h e l i x were v i s i b l e . ) T h e average filament w i d t h i n the a i r - d r i e d gel was f o u n d to be 14.2Â. A f t e r c o r r e c t i n g for the added size due to the P t / C c o a t i n g (39), the c i t r u s p e c t i n filament d i a m e t e r was 5.8 ± 2 A (n = 3 7 , S . D . = 2 Â ) . I n F i g u r e 5 the c i t r u s p e c t i n molecules showed a l e f t - h a n d e d surface s t r i a t i o n o c c u r r i n g every 13Â. T h e surface h e l i x p e r i o d f r o m b o t h t o b a c c o a n d c i t r u s p e c t i n samples was i n agreement w i t h the x - r a y fiber d i f f r a c t i o n measurements (32). I n c o n c l u s i o n , we have d e m o n s t r a t e d t h a t h i g h r e s o l u t i o n T E M is a v a l u a b l e complement to x - r a y fiber diffraction a n a l y s i s a n d c h e m i c a l s t r u c t u r a l e l u c i d a t i o n . Its a p p l i c a t i o n p r o v i d e d i n f o r m a t i o n a b o u t the o r g a n i z a t i o n of p e c t i n i n cell walls a n d i n calcium-free gels. U s i n g freeze-dried s a m ples t h a t were P t / C r e p l i c a t e d , we d e m o n s t r a t e d t o b a c c o p e c t i n filaments i n a gel to be of the same d i a m e t e r as the filaments o n the n o n c u t i n i z e d lower e p i d e r m a l surface of senescing C o k e r 319 tobacco leaves. T h e s e filaments were 7.1 ± 3Â a n d 4.6 ± 4.8Â, respectively, a n d r o u g h l y the same d i a m e t e r , ~ 7Â, as fiber-diffraction modeled c i t r u s p e c t i n (32). R e p l i c a t e d

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F i g u r e 4 A . Freeze-dried, P t / C r e p l i c a t e d gel p r e p a r e d f r o m deesterified t o ­ bacco p e c t i n . ( B a r = ΙΟ,ΟΟΟΑ.)

F i g u r e 4 B . F r e e z e - d r i e d , P t / C r e p l i c a t e d gel prepared f r o m t o b a c c o p e c t i n . T h i s h i g h m a g n i f i c a t i o n image shows two molecules w i t h ~ 13Â left-handed h e l i c a l regions. ( B a r = 100A.)

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F i g u r e 5. A i r - d r i e d , P t / C r e p l i c a t e d gel prepared f r o m c i t r u s p e c t i n . T h i s image features two p e c t i n filaments w i t h left-handed surface s t r i a t i o n s h a v i n g ~ 13Â spacings ( E a c h bar — 25Â.)

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c i t r u s p e c t i n filaments were also f o u n d t o have s i m i l a r diameters, 5.8 ± 2 Â . In a d d i t i o n , we d e m o n s t r a t e d images, for t h e first t i m e , o f the l e f t - h a n d e d surface spacings o f ~ 13Â i n single p e c t i n molecules, i n b o t h freeze-dried t o b a c c o a n d a i r - d r i e d citrus p e c t i n . W e believe t h a t single molecule i m a g i n g c a n c o n t r i b u t e t o a m o r e t h o r o u g h u n d e r s t a n d i n g o f the role o f p e c t i n i n t h e cell w a l l . O u r future efforts w i l l be focused o n p e c t i n gels f o r m e d i n the presence o f c a l c i u m . E v e n t u a l l y , it s h o u l d be possible t o v i s u a l i z e side chains o n p e c t i n a n d determine h o w r h a m n o s e residues i n t h e r h a m n o g a l a c t u r o n a n b a c k b o n e o f t o b a c c o p e c t i n d i s r u p t t h e f o r m a t i o n o f h e l i c a l regions.

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