Chapter 32 Enzyme Excretion During Wood Cell Wall Degradation by Phanerochaete chrysosporium Jean-Paul Joseleau and Katia Ruel Centre de Recherches sur les Macromolécules Végétales, CERMAV-CNRS, B.P. 53X 38041, Grenoble Cedex, France
Wood degradation by Phanerochaete chrysosporium, with the hyphae in contact with, or at a distance from the host cell walls, was examined by electron microscopy with immunocytochemical techniques. An anti-ligninase antibody and antiserum raised against a mixture of cellulases and hemicellulases secreted from the fungus were used. The respective enzymes were localized in intracellular vesicles and seemed to be able to diffuse from the hyphae only when at a short distance from the site of degradation. When excreted from the hyphae the enzymes seemed to be associated with the 1-3, 1-6 β-glucan which forms the sheath secreted during secondary growth of the fungus. The limited distance of migration of the enzymes suggested that a direct contact is needed be tween the wood and the fungal walls for degradation to occur. The propagation of the degradation might take place by an oxygen radical mechanism as revealed by the use of a specific cytochemical method. A m o n g w o o d - r o t t i n g f u n g i the basidiomycete Phanerochaete chrysosporium is able t o degrade b o t h l i g n i n a n d polysaccharides f r o m w o o d cell walls (1). E x a m i n a t i o n o f w h i t e - r o t decayed w o o d at t h e u l t r a s t r u c t u r a l level reveals t h a t several types o f d e g r a d a t i o n can o c c u r (2,3). I n t h e case o f P. chrysosporium a n d i t s a n a m o r p h o u s f o r m (Sporotrichum pulverulentum), t w o p a t t e r n s o f a t t a c k were observed (4). I n one, t h e h y p h a e were t i g h t l y associated w i t h the degraded walls a n d t h i s t y p e o f image can be s a i d t o be " i n c o n t a c t " ( F i g . I A ) . I n the second case, the secondary w a l l was degraded b u t n o h y p h a e c o u l d be observed i n t h e nearby s u r r o u n d i n g s . T h i s t y p e o f d e g r a d a t i o n c a n be described as " a t a d i s t a n c e " ( F i g . I B ) (4). T h e s e t w o extreme aspects o f lysis o f wood cell walls suggest t h a t P. chrysospo rium c a n degrade t h e lignocellulosic c o m p l e x o f the w a l l b y a t least t w o 0097-6156/89/0399-0443$06.00/0 © 1989 American Chemical Society
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F i g u r e 1. T w o extreme aspects o f Populus cell w a l l d e g r a d a t i o n by P. chrysosporium. I A , " i n contact;" I B , "at a distance." ( H = h y p h a ; S i , S2 = w o o d secondary w a l l layers.)
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difTerent b i o c h e m i c a l m e c h a n i s m s . C l a s s i c a l l y , biodégradation of cellulose a n d hemicellulose is ascribed to c e l l u l o l y t i c a n d h e m i c e l l u l o l y t i c enzymes o f the fungus a n d l i g n i n b r e a k d o w n is a t t r i b u t e d to the o x i d a t i v e d e g r a d a t i o n b y l i g n i n - p e r o x i d a s e s (5,7). A l l these enzymes have been s h o w n to be p r o d u c e d b y P. chrysosporium i n v a r y i n g p r o p o r t i o n s , d e p e n d i n g on g r o w t h c o n d i t i o n s (8). T h e i r release b y the h y p h a e at the site of the a t t a c k s h o u l d agree w i t h the p a t t e r n o f w o o d d e g r a d a t i o n s a i d to be " i n c o n t a c t . " H o w e v e r , i t is far m o r e difficult to e x p l a i n the p a t t e r n d e s c r i b e d as " a t a d i s t a n c e , " since i n t h i s m o d e of d e g r a d a t i o n the l y t i c enzymes need to be first t r a n s p o r t e d f r o m the f u n g a l h y p h a e to the w o o d cell w a l l s . T h e y must t h e n diffuse w i t h i n the w a l l , i n order to effect a p a r t i a l a n d l o c a l b r e a k d o w n o f the p o l y s a c c h a r i d e s a n d of l i g n i n . S u c h e n z y m e diffusion i n the c o m p a c t s t r u c t u r e o f the w o o d cell w a l l has never been d e m o n s t r a t e d , a n d does not seem c o m p a t i b l e w i t h the r e l a t i v e l y large size o f these h y d r o l y t i c and o x i d a t i v e enzymes. A n o t h e r p o s s i b i l i t y w h i c h c o u l d e x p l a i n the p a r t i a l d e g r a d a t i o n of the w a l l w o u l d be the i n v o l v e m e n t of n o n - e n z y m a t i c diffusable agents of s m a l l size, w h i c h c o u l d be generated at a c e r t a i n phase of the h y p h a l g r o w t h , or at a c e r t a i n stage o f b i o c h e m i c a l a t t a c k . I n t h i s respect, a c t i v a t e d o x y g e n species have been p o s t u l a t e d as p o t e n t d e g r a d a t i v e e x t r a c e l l u l a r agents, w h i c h c o u l d be p r o d u c e d b y the fungus (9-11). T h i s chapter describes the use of electron microscopy, coupled w i t h i m m u n o c y t o c h e m i c a l techniques, to investigate the a b i l i t y of l i g n o l y t i c enzymes f r o m P. chrysosporium to diffuse inside the w o o d cell w a l l . Materials and
Methods
Plant Material. W o o d samples were t a k e n f r o m a 20-year-old aspen tree (Populus tremula) harvested i n F r a n c e . W o o d wafers (4 χ 20 x 50 m m ) were degraded b y the w i l d t y p e s t r a i n K 3 of the w h i t e rot fungus P. chrysospo rium at the S T F I ( S t o c k h o l m , Sweden) i n the l a b o r a t o r y o f D r . K . - E . Eriksson. Preparation of Antisera. A n t i s e r a directed against the crude enzymes m i x t u r e secreted b y P. chrysosporium a n d c u l t i v a t e d i n a f e r m e n t o r o n cel lulose, were raised i n r a b b i t s . I m m u n o g l o b u l i n s G ( = I g G ) were p u r i f i e d at the I n s t i t u t e P a s t e u r ( L y o n ) a n d used for i m m u n o l a b e l i n g . S e c o n d a r y goat a n t i r a b b i t g o l d m a r k e r was f r o m S i g m a . T h e a n t i - l i g n i n a s e a n t i s e r u m was raised i n r a b b i t f r o m a p u r i f i e d ligninase (gift of D r . E . O d i e r , I N A , Paris-Grignon, France). Tissue Preparation for Electron Microscopy. T i s s u e s were fixed i n 2 % p a r a f o r m a l d e h y d e , 2 . 5 % g l u t a r a l d e h y d e i n p h o s p h a t e buffer (0.1 M , p H 7.4) a n d 0 . 0 2 % p i c r i c a c i d . T h e y were then d e h y d r a t e d i n g l y c o l m e t h a c r y l a t e m o n o m e r a n d e m b e d d e d i n g l y c o l m e t h a c r y l a t e ( G M A ) (24). Immunocytochemical Labeling. A n t i b o d i e s were used as p o s t - e m b e d d i n g m a r k e r s . Sections o f decayed w o o d were first i n c u b a t e d o n a d r o p o f T B S ( T r i s - p h o s p h a t e s a l i n e buffer 0.1 M , p H 7.4, N a C l 0.15 M or 0.5 M ) , g l y c i n e 0.15 M. A f t e r r i n s i n g i n T B S , they were floated o n a d r o p of 1% T B S B S A (bovine s e r u m a l b u m i n ) (or n o n - i m m u n e goat serum) before t r e a t i n g
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either w i t h I g G a n t i - c r u d e enzymes ( 1 9 ^ g / m l d i l u t e d i n T B S - B S A ) (or T B S / n o r m a l goat s e r u m = T B S / N G S ) , or a n t i - l i g n i n a s e a n t i s e r u m d i l u t e d 1:250 i n the same buffer, for 60 m i n at r o o m t e m p e r a t u r e . T h e secondary a n t i s e r a labeled w i t h g o l d (10 n m i n d i a m e t e r ) was a goat a n t i r a b b i t a n tisera purchased f r o m Janssen ( P h a r m a c e u t i c a , Beerse, B e l g i u m ) . It was d i l u t e d 1:30 i n T B S / N G S . T h e sections were e x a m i n e d o n a P h i l i p s 400 Τ electron microscope w i t h o u t a n y c o u n t e r s t a i n i n g . Immunocytochemical Controls. a. S u b s t i t u t i o n of the p r i m a r y a n t i b o d y w i t h p r e i m m u n e r a b b i t s e r u m IgG fraction. b . T r e a t m e n t o f section w i t h g o a t - a n t i r a b b i t g o l d - l a b e l l e d secondary a n t i b o d y alone, o m i t t i n g the p r i m a r y a n t i b o d y step. c. L a b e l i n g w i t h a n t i s e r a p r e a d s o r b e d w i t h t h e i r respective antigens. E q u a l volumes o f a n t i - c r u d e enzymes a n d of the e n z y m a t i c e x t r a c t , o r , a n t i - l i g n i n a s e and p u r e ligninase, were i n c u b a t e d 1 h before use. Carbonyl Groups Labeling. T h i s was done under the u s u a l c o n d i t i o n s of P A T A g s t a i n i n g (periodic a c i d , t h i o c a r b o h y d r a z i d e , silver proteinate) reac t i o n s (2) o m i t t i n g the p e r i o d a t e o x i d a t i o n step ( = T A g ) , as described i n (21). F e n t o n ' s reagent was prepared a n d a p p l i e d as described i n (18). Results and Discussion "Diffusion" of the Fungal Glycohydrolases. A crude e n z y m e m i x t u r e f r o m P. chrysosporium was o b t a i n e d b y a m m o n i u m sulfate p r e c i p i t a t i o n f r o m a c u l t u r e g r o w n o n cellulose and m a i n t a i n e d i n p r i m a r y g r o w t h c o n d i t i o n s . T h i s e n z y m e m i x t u r e , w h i c h was used for p r e p a r i n g a p o l y c l o n a l a n t i s e r u m , c o n t a i n e d inter alia p r e d o m i n a n t l y endo- and exoglucanases, w i t h o n l y traces o f hemicellulases as evidenced b y t h e i r a c t i o n o n the c o r r e s p o n d i n g s u b strates. N o l i g n i n peroxidase a c t i v i t y c o u l d be detected ( K . - E . E r i k s s o n , p e r s o n a l c o m m u n i c a t i o n ) . T h e i m m u n o g l o b u l i n s ( I g G ) raised i n r a b b i t were first used to detect the site of secretion of the enzymes i n the f u n g a l cells. T h e p r i m a r y a n t i b o d i e s were l o c a l i z e d w i t h a goat a n t i r a b b i t sec o n d a r y a n t i b o d y a d s o r b e d to c o l l o i d a l g o l d . T h e d i a m e t e r of g o l d p a r t i c l e s was 10 n m . C o n t r o l s w i t h the anti-glycohydrolases I g G , first i n c u b a t e d w i t h the e n z y m i c e x t r a c t before b e i n g a p p l i e d i n t h i n sections, showed n o l a b e l i n g ( F i g . 2 B ) . O t h e r controls p e r f o r m e d , i.e., replacement of the p r i m a r y a n t i b o d y b y n o r m a l r a b b i t s e r u m or p r e i m m u n e s e r u m , suppression o f the first step c o r r e s p o n d i n g t o the p r i m a r y a n t i b o d y , or l a b e l i n g o f uninfected w o o d s p e c i m e n , were also negative. T h e h y p h a e p h o t o g r a p h e d i n a h i g h l y decayed w o o d e x h i b i t e d several aspects of their c y t o p l a s m i c content. T h e l o c a l i z a t i o n of the glycohydrolases v a r i e d a c c o r d i n g l y as s h o w n i n F i g u r e 2. In F i g u r e 2 A , they are l o c a l i z e d i n t r a c e l l u l a r ^ i n s m a l l dense vesicles (arrows). In F i g u r e 2 C they appeared c o n c e n t r a t e d a l o n g the p l a s m a l e m m a a n d dispersed t h r o u g h the c y t o p l a s m ( a r r o w s ) . In F i g u r e 3 A , they have been secreted out of the h y p h a ( a r r o w s ) . T h e s e observations o f the presence o f a n i n t r a c e l l u l a r c e l l u l o l y t i c a c t i v i t y
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Figure 2. Labeling with anti-crude enzyme mixture. A and Β show the specificity of the antibodies compared to the preimmune-treated control; C, plasmatic and cytoplasmic localization of the enzymes. No labeling in the fungal wall. (Pm, plasmalemma; W, hyphal wall.)
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i n the h y p h a agree w i t h the results of M u r m a n i s et al. (12). I n zones of less advanced d e g r a d a t i o n ( F i g . 3 B ) , h y p h a e " i n c o n t a c t " w i t h p a r t i a l l y degraded w o o d cell walls h a d no enzymes i n their o w n w a l l s , b u t some glycohydrolases c o u l d be observed p e n e t r a t i n g a short distance i n t o the w o o d cell w a l l . T h e g o l d p a r t i c l e s i n w o o d are o n l y seen i n lightened areas where w o o d is a l r e a d y degraded. Nevertheless, this was a n i n d i c a t i o n t h a t some glycohydrolases c o u l d pass t h r o u g h the f u n g a l cell w a l l a n d penetrate a short distance i n t o w o o d cell w a l l s . In areas of the degraded w o o d where no h y p h a e are v i s i b l e i n the s u r r o u n d i n g s ( p a t t e r n "at a d i s t a n c e " ) , no g o l d particles were evident i n the decayed w o o d cell w a l l s . T h i s suggested t h a t i n t h i s case the enzymes r e m a i n associated w i t h the h y p h a e (or the h y p h a l sheath). "Diffusion" of Lignin-peroxidase (Ligninase). A n anti-ligninase antiserum was raised i n a r a b b i t u s i n g a p u r i f i e d ligninase f r a c t i o n (13). In t h i s s t u d y , the g o l d l a b e l i n g was p e r f o r m e d w i t h the t o t a l a n t i s e r u m . A s can be seen i n F i g u r e 4, g o l d particles were l o c a l i z e d i n the c y t o p l a s m , a l o n g the p l a s m a l e m m a ( F i g . 4 A ) a n d i n t o the h y p h a l w a l l i n F i g u r e 4 C . T h i s l o c a l i z a t i o n o f l i g n i n a s e differs f r o m t h a t r e p o r t e d by M e s s n e r et al. (15). Some m i n o r l a b e l i n g was also present i n t r a c e l l u l a r l y i n some cells ( F i g . 4 A ) as described b y G a r c i a et al. (16). Interestingly, some hyphae d i d not show any affinity for the ligninase a n t i b o d y ; these m i g h t correspond to those h y p h a e w h i c h d i d not secrete ligninase (14). [ F i g . 4 B corresponds to a c o n t r o l , where the a n t i b o d y was p r e i n c u b a t e d w i t h the ligninase before b e i n g a p p l i e d o n the section.] In a l l cases e x a m i n e d , a n d regardless of the relative s i t u a t i o n of the h y p h a t o the w o o d cell w a l l , no e x t r a c e l l u l a r a c c u m u l a t i o n of l i g n i n a s e was detectable at the site of d e g r a d a t i o n . However, a n o r i e n t e d secretion of ligninase o u t w a r d s , across the hyphae w a l l , was observed i n F i g u r e 4 D . In t h i s p h o t o g r a p h the p h y s i o l o g i c a l state of the h y p h a c o u l d not be ascert a i n e d a n d i t is therefore difficult to correlate the e x c r e t i o n of ligninase to a given state of the h y p h a . However, it c o u l d be possible t h a t the e x c r e t i o n o f ligninase c o u l d o c c u r i n s u b l e t h a l h y p h a e i n w h i c h the c y t o p l a s m i c m e m brane c o u l d have a m o d i f i e d p e r m e a b i l i t y . In F i g u r e 4 E , there is a burst of l i g n i n peroxidase outside a h y p h a w h i c h is o b v i o u s l y d e a d . F r o m the above results, a n d i n agreement w i t h recent reports (15,16), i t appears t h a t P. chrysosporium does not secrete the b u l k of its l i g n o l y t i c enzymes d u r i n g n a t u r a l d e g r a d a t i o n of w o o d ; t h i s is i n contrast to its secretion i n b a t c h cultures, w h i c h bears l i t t l e resemblance to the n a t u r a l d e g r a d a t i o n o f w o o d tissues. These findings, however, provide no e x p l a n a t i o n for the d e g r a d a t i o n w h i c h takes place at some distance f r o m the h y p h a e . If enzymes are not d i r e c t l y i n v o l v e d i n t h i s process, the diffusion of n o n - e n z y m a t i c agents, such as r a d i c a l cations or a c t i v a t e d oxygen species m a y be o c c u r r i n g (16,18). In t h i s r e g a r d , i t is k n o w n t h a t oxygen species p r o d u c e d v i a h y d r o g e n peroxide, e.g., the h y d r o x y l r a d i c a l ( * O H ) , are able to a t t a c k b o t h the l i g n i n a n d polysaccharides (9,19,20). Action
of Hydroxyl
Radicals
on Wood Cell
Walls.
In order to investigate
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Figure 3. Labeling with anti-crude enzyme mixture. A , some labeling was observed on the electron-dense sheath (arrow). No post-staining. In B, some gold markers were localized on the outer part of the degraded S layer. No labeling of the hyphal wall. (Pm, plasmalemma; W, hyphal wall.) 2
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F i g u r e 4. L a b e l i n g w i t h a n t i - l i g n i n a s e . 4 A : M o s t of the l a b e l i n g was e v i d e n t i n the p l a s m a t i c area a n d also i n the w a l l . T h e l a b e l i n g is s t i l l seen o n the e m p t y hyphae ( 4 A a n d 4 C ) . 4 B corresponds t o a c o n t r o l ; the a n t i b o d y was p r e i n c u b a t e d w i t h the ligninase before b e i n g a p p l i e d o n the section. 4 D : A g r o u p of h y p h a e at different p h y s i o l o g i c a l states. In some h y p h a e ligninase was clearly s h o w n i n the w a l l a n d crossing it o u t w a r d s (arrows). 4 E : A n e m p t y h y p h a is clearly e x c r e t i n g the b u l k of its ligninase (arrows). ( P m = plasmalemma; W = hyphal wall; H = hypha.)
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the possible d e g r a d a t i o n of w o o d c o m p o n e n t s v i a * O H , s o u n d w o o d s a m p l e s were s u b m i t t e d to the a c t i o n o f Fen t o n ' s reagent w h i c h is a g o o d s y s t e m for p r o d u c i n g h y d r o x y l r a d i c a l s . P r e v i o u s results (18) showed t h a t a c t i v a t e d o x y g e n species generated i n situ created p a t t e r n s o f d e g r a d a t i o n h i g h l y c o m p a r a b l e w i t h those created b y the fungus. T h i s o x i d a t i v e a c t i o n o f the fungus c a n be v i s u a l i z e d w i t h electron m i c r o s c o p y i n degraded w o o d s a m ples, u s i n g a specific m e t h o d designed to detect the c a r b o n y l a n d c a r b o x y l groups created i n decayed w o o d b y the fungus (21). F o l l o w i n g c o m p a r i s o n t o s o u n d w o o d ( F i g s . 5 A a n d 5 B ) , i t was c l e a r l y e v i d e n t t h a t degraded w o o d h a d undergone o x i d a t i o n . T h i s o x i d a t i v e p r o cess c o u l d take place o n b o t h polysaccharides a n d l i g n i n (22,23), a n d c o u l d therefore represent a general d e g r a d a t i o n m e c h a n i s m o f the w o o d cell w a l l polymers. Conclusion D i f f u s i o n of enzymes f r o m the f u n g a l cells is c o m p l i c a t e d by the fact t h a t h y p h a e behave differently d e p e n d i n g u p o n t h e i r p h y s i o l o g i c a l state. I m m u n o c y t o c h e m i s t r y allowed the v i s u a l i z a t i o n of p o l y s a c c h a r i d e - d e g r a d i n g enzymes a n d l i g n i n peroxidases d u r i n g t h e i r secretion b y the fungus. P e n e t r a t i o n o f cellulases, hemicellulases a n d l i g n i n a s e was l i m i t e d a n d no g o l d
F i g u r e 5. C a r b o n y l group l a b e l i n g . A p p l i c a t i o n of the T A g sequence. 5 A : C o n t r o l = T A g o n s o u n d w o o d . 5 B : s i l v e r g r a i n deposits c o r r e s p o n d to the c a r b o n y l groups created b y the fungus. ( M L + P W = m i d d l e l a m e l l a + p r i m a r y w a l l ; S i a n d S2 = outer a n d m i d d l e layers of the secondary w a l l , respectively.)
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labeling could be seen in sound wood. This means that enzymes invaded the wood cell walls only in places where a predegradation had already oc curred. The oxidative action of Fenton's reagent which could be followed by specific cytochemistry suggested that activated oxygen species could partic ipate in the propagation of the degradation initiated by lignin peroxidase. This might be an explanation of the type of decay "at a distance." Acknowledgments Thanks are expressed to Dr. K.-E. Eriksson (STFI, Stockholm, Sweden) for the gift of the enzyme extract and inoculation of the wood samples; to Professor R. Guinet (Institut Pasteur, Lyon Lentilly, France) for the preparation of the IgG directed against the crude protein extracted; and to Dr. E . Odier (INA P.G., France) for providing the anti-ligninase antiserum. Literature Cited 1. Eriksson, K.-E. Pure Appl. Chem. 1981, 53, 33-43. 2. Ruel, K.; Barnoud, F. In Biosynthesis and Biodegradation of Wood Components; Higuchi, T . , Ed.; Academic: New York, 1985, p. 441. 3. Otjen, L.; Blanchette, R. A. Can. J. Bot. 1982, 60, 2770-94. 4. Ruel, K.; Joseleau, J . P. Symbiosis 1986, 2, 355-61. 5. Murmanis, L.; Highley, T.; Palmer, J . G . Holzforschung 1984, 38, 1118. 6. Tien, M.; Kirk, T . K. Science 1983, 221, 661-63. 7. Eriksson, K.-E.; Wood, T . M . In Biosynthesis and Biodegradation of Wood Components; Higuchi, T . , Ed.; Academic: New York, 1985; p. 469. 8. Ander, P.; Eriksson, K . - E . Physiol. Plant. 1977, 41, 239-248. 9. Nakatsubo, F.; Reid, I. D.; Kirk, T . K. Biochem. Biophys. Res. Com mun. 1981, 102, 484-91. 10. Forney, L. J.; Reddy, Α.; Tien, M.; Ausi, S. D. J. Biol. Chem. 1982, 257, 11455-62. 11. Schoemaker, Η. E.; Harvey, P. J.; Bowen, R. M.; Palmer, J. M . FEBS Lett. 1985, 183, 7-12. 12. Murmanis, L.; Highley, T . L.; Palmer, J . G. Sci. Technol. 1987, 21, 101-09. 13. Tonon, F.; Odier, E.; Asther, M.; Lesage, L.; Corrieu, G . In Lignin En zymic and Microbial Degradation; E . Odier, Ed.; INRA Publications: 1987; pp. 165-70. 14. Keyser, P.; Kirk, T . K.; Zeikus, J . G . J. Bact. 1978, 135, 3, 790-97. 15. Messner, K.; Strebotnik, E . ; Erler, G.; Foisner, R.; Pettersson, B.; Stachelberger, H. In Lignin Enzymic and Microbial Degradation; Odier, E . , Ed.; INRA Publications: 1987; 243-48. 16. Garcia, S.; Latgé, J . P.; Prevost, M . C.; Leisola, M . Appl. Environ. Microbiol. 1987, 53, 2384-87. 17. Gupta, D. P.; Heale, J . B. J. Gen. Microbiol. 1971, 63, 163-73. 18. Ruel, K.; Joseleau, J . P. Food Hydrocolloids 1987, 1, 515-17. 19. Koenigs, J . W. Wood Fibers 1974, 6, 66-79.
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20. Weldock, D. J.; Parsons, B. J.; Phillips, G . O.; Thomas, B. In Cellulose and its Derivatives; Kennedy, J . F., Ed.; John Wiley and Sons: New York, 1985; 46, pp. 503-10. 21. Joseleau, J . P.; Ruel, K. Biol. Cell. 1985, 53, 61-66. 22. Isbell, H. S.; Frush, H. L. Carbohydr. Res. 1987, 161, 181-93. 23. Gilbert, B. C.; King, D. M.; Thomas, C. B. Carbohydr. Res. 1984, 125, 217-35. 24. Spaur, C. R.; Moriarty, G . C. J. Histochem. Chem. 1977, 25, 163-74. RECEIVED March 17, 1989
