Chapter 31
Ultrastructural Localization of Lignocellulose -Degrading Enzymes 1
1
1
2
I. M. Gallagher , M. A. Fraser , C. S. Evans , P. T. Atkey , and D. A. Wood 2
1
School of Biological Sciences, Thames Polytechnic, London SE18 6PF, England AFRC Institute of Horticultural Research, Littlehampton BN16 3PU, England
2
Transmission electron microscopy of immuno-gold labelled sections was used to show the localisation of the ligninolytic enzymes, lignin-peroxidase and laccase in ultrathin sections of hyphae of the white-rot fungus Coriolus versicolor. Both enzymes were localized in the fungal cell walls and mucilage layers of both generative and skeletal hyphae, whereas ligninase but not laccase was also evident adjacent to the plasma membrane. The localization of these enzymes was the same in hyphae grown in culture and in sections of beech heartwood infected with C. versicolor. Control experiments showed that no labelling was detected in the absence of primary antibodies, or when antibodies to animal or plant enzymes were substituted but antibodies to fungal proteins from different species were labelled in sections of C. versicolor. The source of antigenicity in these sections was investigated. B a s i d i o m y c e t e f u n g i are the m a j o r organisms responsible for biodégradation of w o o d , w i t h w h i t e - r o t fungi able t o degrade a l l components o f the w o o d cell w a l l . E n z y m e s w h i c h p a r t i a l l y degrade l i g n i n , l i g n i n - p e r o x i d a s e s , were first isolated f r o m Phanerochaete chrysosponum (1-2) a n d more recently f r o m other w h i t e - r o t fungi i n c l u d i n g Coriolus versicolor (3). These enzymes appear t o be s i m i l a r i n a l l w h i t e - r o t f u n g i investigated, a l l c o n t a i n i n g a heme p r o s t h e t i c group a n d r e q u i r i n g trace a m o u n t s o f h y d r o g e n peroxide t o effect t h e b r e a k d o w n o f the l i g n i n p o l y m e r (4-5). T h e p o l y p h e n o l oxidase, laccase, p r o d u c e d as a n e x t r a c e l l u l a r enzyme b y C. versicolor, has been w e l l d o c u m e n t e d as a p o l y m e r i z i n g enzyme b u t recent work has s h o w n t h a t under c e r t a i n c o n d i t i o n s i t c a n also effect d e p o l y m e r i z a t i o n o f l i g n i n (6-8). 0097-6156/89/0399-0426$06.00/0 © 1989 American Chemical Society
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M o r p h o l o g i c a l d a t a o n t h e patterns o f d e g r a d a t i o n o f w o o d cell w a l l s after i n f e c t i o n b y w h i t e - r o t f u n g i showed t h a t i n a d d i t i o n t o w h i t e - r o t s c a u s i n g a l o c a l i z e d d e g r a d a t i o n , characterized b y a deep erosion t r o u g h i n the secondary w a l l o f the w o o d cell, a progressive t h i n n i n g o f the S2 layer of the secondary w a l l also o c c u r r e d (9-12). I d e n t i f i c a t i o n o f o s m i o p h i l i c p a r ticles i n sections o f d e c a y i n g w o o d led t o t e n t a t i v e suggestions t h a t these were either e n z y m e molecules i n v o l v e d i n w o o d decay or t h e p r o d u c t s of lignocellulosic d e g r a d a t i o n (12-13). Studies o f t h e b i o c h e m i c a l m e c h a n i s m of l i g n i n - p e r o x i d a s e have suggested t h a t i t is n o t always essential t h a t t h e e n z y m e s h o u l d be i n direct contact w i t h i t s substrate t o effect l i g n i n b r e a k d o w n . T h e release o f diffusible r a d i c a l cations i n p h e n o l i c substrates c a n enable d e g r a d a t i o n o f t h e l i g n i n p o l y m e r t o proceed at some distance f r o m the e n z y m e , a n d f u n g a l h y p h a (14 ). W h i t e - r o t f u n g i secrete a p o l y s a c c h a r i d e mucilage a r o u n d the h y p h a e w h i c h m a y f u n c t i o n as a m a t r i x for e n z y m e i m m o b i l i z a t i o n a n d enable p r o d u c t s o f reactions t o be r e t a i n e d close t o t h e h y p h a e for a b s o r p t i o n (15; E v a n s , C . S., u n p u b l i s h e d d a t a ) . Recently, significant advances were m a d e i n the field o f i m m u n o - l a b e l l i n g w i t h electron m i c r o s c o p y as a m e t h o d o f l o c a l i z i n g proteins a n d other molecules w i t h i n tissue sections, u s i n g c o l l o i d a l g o l d as a m a r k e r o f a n t i b o d y - a n t i g e n i n t e r a c t i o n s (16). I n order t o i d e n tify the site o f a c t i o n o f l i g n i n - d e g r a d i n g enzymes i n w o o d decay, c o l l o i d a l g o l d i m m u n o l o c a l i z a t i o n procedures were used t o locate the e x t r a c e l l u l a r enzymes, laccase a n d l i g n i n - p e r o x i d a s e , f r o m the w h i t e - r o t fungus C. versicolor c u l t u r e d i n beech h e a r t w o o d a n d i n m a l t agar. Methods Organism. Coriolus versicolor s t r a i n 2 8 A P R L ( B u i l d i n g Research E s t a b l i s h m e n t , P r i n c e s R i s b o r o u g h L a b o r a t o r y , A y l e s b u r y , B u c k s . , U . K . ) was m a i n t a i n e d o n a s o l i d m e d i u m o f 3 % ( w / v ) m a l t e x t r a c t , 2 % ( w / v ) agar at 2 0 ° C , or o n n i t r o g e n - l i m i t e d n u t r i e n t m e d i u m (17) solidified w i t h 2 % agar. Growth ofC. versicolor on Wood. S a m p l e s o f beech h e a r t w o o d (Fagus sylvatica) were added t o c u l t u r e plates of C. versicolor after 7 d . g r o w t h , u s i n g the B r a v e r y m i n i a t u r e woodblock technique (12). P l a t e cultures were covered w i t h a 1 m m mesh 6 0 m m diameter sterile n y l o n n e t . S t e r i l e sections of beech (30 x 10 x 3 m m ) were placed aseptically onto the net a n d c u l t u r e d at 2 0 ° C for 4 weeks. T h e e x t e r n a l g r o w t h o f m y c e l i u m was removed a n d the w o o d block c u t i n t o segments o f a p p r o x i m a t e l y 3 x 1 x 1 m m for p r e p a r a t i o n for electron microscopy. U n i n f e c t e d w o o d samples were prepared i n a similar manner. Immunogold Labelling. Tissues were fixed i n excess g l u t a r a l d e h y d e ( 2 . 5 % w / v ) i n l O O m M s o d i u m cacodylate buffer ( p H 7.2) for 3 h , washed twice i n t h e same buffer, followed b y d e h y d r a t i o n i n a graded e t h a n o l series. S a m p l e s were i n f i l t r a t e d w i t h L . R . W h i t e h a r d grade resin ( L o n d o n R e s i n C o . L t d ) . T h i n sections were cut w i t h a n L K B U l t r a m i c r o t o m e II u s i n g a d i a m o n d knife a n d collected o n copper grids (200 mesh). T h e i m m u n o g o l d l a b e l l i n g procedure followed the technique o f B e r g m a n (18). Sections were
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PLANT C E L L W A L L P O L Y M E R S
etched i n 5 % H 2 O 2 for 5-10 m i n , washed twice i n P B S ( 2 0 m M p h o s p h a t e buffer at p H 7.4 a n d 0.9% N a C l ) c o n t a i n i n g 0 . 1 % T w e e n 20, i n c u b a t e d for l h i n r a b b i t anti-laccase a n t i s e r u m or r a b b i t 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 1:100 ( v / v ) i n P B S c o n t a i n i n g 0 . 1 % T w e e n 20 a n d 1% b o v i n e s e r u m a l b u m e n ( B S A ) , washed twice i n P B S c o n t a i n i n g 0 . 1 % T w e e n 20, i n c u b a t e d for l h i n goat a n t i - r a b b i t I g G conjugated to l O n m g o l d particles ( S i g m a ) , d i l u t e d 1:20 i n P B S c o n t a i n i n g 1% B S A a n d 0 . 1 % T w e e n 20, washed twice i n d i s t i l l e d water a n d air d r i e d . Post-staining Procedure. I m m u n o g o l d l a b e l l e d sections were p o s t - s t a i n e d i n s a t u r a t e d aqueous u r a n y l acetate for 15 m i n a n d lead c i t r a t e for 10 m i n (19) . Transmission Electron Microscopy. T r a n s m i s s i o n electron m i c r o s c o p y was p e r f o r m e d o n a J e o l 100S electron microscope o p e r a t i n g at 8 0 k V . Production of Antibodies. Laccase A was isolated f r o m cultures of C. versicolor a n d p u r i f i e d b y a m o d i f i e d procedure of Fahraeus a n d R e i n h a m m e r (20) . T h e c r i t e r i o n for p u r i t y was a single b a n d o n a h e a v i l y l o a d e d S D S p o l y a c r y l a m i d e electrophoresis gel. A n t i b o d i e s were raised i n r a b b i t s i n response to three intravenous injections each of 2 m g . of laccase A w i t h F r e u n d s a d j u v a n t , at 2 weekly intervals. B l o o d was removed 7d after the last i n j e c t i o n a n d s e r u m collected after c o a g u l a t i o n of the b l o o d cells. T h e a n t i b o d y to laccase A was purified b y affinity c h r o m a t o g r a p h y o n C N - B r a c t i v a t e d Sepharose 4 B . Laccase (15mg) was b o u n d to a c o l u m n of 3 . 5 m l v o l u m e i n a c o u p l i n g buffer of 0 . 1 M N a H C 0 a n d 0 . 5 M N a C l . T h e r e m a i n i n g a c t i v e groups o n the c o l u m n were blocked w i t h 0 . 2 M g l y c i n e , before excess adsorbed p r o t e i n was removed w i t h the c o u p l i n g buffer, res u l t i n g i n 9 5 % c o u p l i n g of the laccase to the c o l u m n . C r u d e a n t i s e r a was b o u n d to the laccase o n the c o l u m n , washed w i t h buffer of 0 . 1 M Na2HP04 a n d 0 . 5 M N a C l , before e l u t i o n w i t h 0 . 2 M g l y c i n e - H C l a n d 0 . 5 M N a C l . T h e purified a n t i b o d y was used as the p r i m a r y a n t i b o d y to laccase A . L i g n i n - p e r o x i d a s e , w h i c h oxidised v e r a t r y l a l c o h o l to v e r a t r a l d e h y d e , was isolated f r o m cultures of Phanerochaete chrysosporium b y the m e t h o d of T i e n a n d K i r k (1). It h a d a m o l e c u l a r weight of 4 4 K d on p o l y a c r y l a m i d e gels, i n the presence of s o d i u m dodecyl s u l p h a t e . A n t i b o d i e s to l i g n i n peroxidase were raised i n r a b b i t s as described for laccase A . 3
Results T h e t i t r e of antibodies i n laccase antisera was 1:16 as measured b y the i m m u n o d i f f u s i o n technique of O u c h t e r l o n y (21). These a n t i b o d i e s were effective i n h i b i t o r s o f catechol oxidase a c t i v i t y , w i t h 100/il of a n t i s e r a red u c i n g a c t i v i t y b y 9 5 % , i n a n assay m i x t u r e of 3 m l o f 0 . 1 M catechol i n 0 . 1 M acetate buffer at p H 5 as described b y E v a n s (7). T a b l e I shows the effect of a d d i n g different volumes of a n t i s e r a to the assay m i x t u r e . I n h i b i t i o n of enzyme a c t i v i t y was not used to assess the a n t i b o d y antigen reaction of l i g n i n - p e r o x i d a s e , as the a d d i t i o n of c o n t r o l s e r u m to e n z y m e reaction m i x t u r e s increased the p H above the p H specificity of the
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T a b l e I. T h e effect of laccase a n t i s e r u m o n e n z y m e a c t i v i t y . T h e r e a c t i o n m i x t u r e c o n t a i n e d 3 m l o f 0.1 M catechol i n 0.1 M acetate buffer at p H 5, 5/ig laccase a n d 0-100/d o f s e r u m V o l u m e o f a n t i s e r a (μ\) a d d e d : Laccase a c t i v i t y (OD440 catechol o x i d a t i o n m i n
- 1
0
10
20
50
100
0.21
0.18
0.15
0.04
0.02
)
e n z y m e , w h i c h has a n o p t i m u m at p H 2.9 a n d is i n h i b i t e d above p H 4 (4). C r o s s r e a c t i v i t y between a n t i b o d y a n d antigen was measured b y i m m u n o p r e c i p i t a t i o n at a d i l u t i o n of 1:16 o f a n t i s e r u m (21). B o t h a n t i b o d i e s showed specificity i n r e a c t i n g w i t h t h e i r respective antigens as d e t e r m i n e d b y W e s t ern b l o t t i n g . T h e d i s t r i b u t i o n of laccase i n h y p h a e o f C. versicolor g r o w n i n beech h e a r t w o o d for 4 weeks was c o m p a r e d w i t h t h a t of h y p h a e c u l t u r e d o n m a l t agar. B o t h cultures gave p o s i t i v e tests for laccase w h e n t r e a t e d w i t h g u a i a c o l , p r o d u c i n g y e l l o w / b r o w n colorations (22). Sections o f h y p h a e f r o m b o t h agar a n d w o o d cultures of C. versicolor were treated w i t h either laccase a n t i s e r u m or purified laccase I g G . N o difference i n the p a t t e r n of l a b e l l i n g was observed w h e n crude antisera was used c o m p a r e d w i t h p u r i fied I g G . F i g u r e 1 showed t h a t hyphae g r o w n o n m a l t agar h a d l a b e l l i n g w h i c h was r e s t r i c t e d to the h y p h a l cell w a l l a n d m u c i l a g e layer, w i t h l i t t l e c y t o p l a s m i c l a b e l l i n g a n d a v e r y low level of b a c k g r o u n d l a b e l l i n g . T h e r e was no l a b e l associated w i t h the p l a s m a m e m b r a n e as seen i n F i g u r e 2, a n d the m a j o r site o f l a b e l l i n g was the cell w a l l ( i n most sections the thickness of the mucilage was so s m a l l as t o be i n d i s t i n g u i s h a b l e f r o m the cell w a l l ) . H y p h a e i n sections of infected w o o d showed s i m i l a r m o r p h o l o g y to t h a t of h y p h a e g r o w n o n agar. A s i m i l a r p a t t e r n of l a b e l l i n g was also observed, w i t h most g o l d particles a t t a c h e d to the cell w a l l a n d m u c i l a g e layers a n d m i n i m a l l a b e l l i n g i n the c y t o p l a s m ( F i g u r e s 3 a n d 4). A t higher m a g n i f i c a t i o n i t was seen t h a t mucilage separated f r o m the h y p h a l w a l l was also l a b e l l e d , as was the secondary w a l l of the w o o d cell w a l l . T h e r e was a m o d e r a t e level o f l a b e l i n the S2 w a l l layer whereas the m i d d l e l a m e l l a a n d decayed m a r g i n s of the w a l l were not l a b e l l e d . Sections o f u n i n f e c t e d beech h e a r t w o o d showed no b a c k g r o u n d l a b e l l i n g of cell walls i n d i c a t i n g t h a t there was no non-specific b i n d i n g of the laccase a n t i b o d y t o w o o d cell w a l l c o m p o n e n t s ( F i g . 5). Sections of C. versicolor h y p h a e g r o w n o n N - l i m i t e d n u t r i e n t agar were also treated w i t h antibodies to l i g n i n - p e r o x i d a s e p u r i f i e d f r o m P. chrysospo rium. C r o s s reactive m a t e r i a l was detected i n the cell w a l l a n d m u c i l a g e of the h y p h a e as s h o w n i n F i g u r e 6. T h e r e was a c o n s i d e r a b l y lower level of l a b e l l i n g i n the c y t o p l a s m . T h e l a b e l w i t h i n the w a l l o f generative h y phae was l o c a l i z e d i n t o two d i s t i n c t layers w h i c h was especially o b v i o u s i n the thickened w a l l a n d mucilage layer, as s h o w n i n F i g u r e 7. T h i s double layer o f l i g n i n - p e r o x i d a s e was s i t u a t e d adjacent to the p l a s m a m e m b r a n e a n d o n the outer surface of the w a l l w i t h i n the m u c i l a g e layer ( F i g u r e 8). In the skeletal h y p h a e there was intense l a b e l l i n g of the thicker cell w a l l
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F i g u r e 1. Single h y p h a of C. versicolor grown on m a l t agar, labelled w i t h r a b b i t anti-laccase antibodies, localized w i t h g o a t - a n t i r a b b i t g o l d c o n j u gate. T h e label is restricted to the h y p h a l w a l l , w i t h l i t t l e label i n the c y t o p l a s m . M a g n i f i c a t i o n χ 16,000.
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F i g u r e 2. H i g h e r m a g n i f i c a t i o n of laccase-labelling i n the h y p h a l w a l l a n d mucilage layer. N o l a b e l is seen on the p l a s m a m e m b r a n e or w i t h i n the c y t o p l a s m . M a g n i f i c a t i o n χ 59,500.
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POLYMERS
F i g u r e 3. Beech h e a r t w o o d , decayed for 4 weeks w i t h C. versicolor, s h o w i n g t h i n n i n g of the secondary w a l l w i t h enlargement of the c a v i t y at the cell corner (the p r o t r u d i n g ends of the m i d d l e l a m e l l a can be seen). A single h y p h a close to the w o o d cell w a l l shows where the secondary w a l l has been degraded. L a b e l for laccase is seen u n i f o r m l y d i s t r i b u t e d i n the secondary w a l l . M a g n i f i c a t i o n χ 12,600.
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F i g u r e 4. H i g h e r m a g n i f i c a t i o n of h y p h a l w a l l i n F i g u r e 3 s h o w i n g l a b e l l i n g of the w a l l a n d mucilage layer. M a g n i f i c a t i o n χ 66,500.
433
intense
F i g u r e 5. A single h y p h a f r o m an agar g r o w n c u l t u r e of C. versicolor s h o w i n g an absence of label after replacing p r i m a r y antisera w i t h r a b b i t I g G g l o b u l i n . M a g n i f i c a t i o n χ 19,800.
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F i g u r e 6. H y p h a e f r o m N - l i m i t e d nutrient agar g r o w n cultures of C. versi color s h o w i n g (a) a thick walled skeletal h y p h a a n d (b) a t h i n walled gen erative h y p h a labelled for lignin-peroxidase. L a b e l is seen t h r o u g h o u t the h y p h a l walls w i t h l i t t l e l a b e l l i n g i n the c y t o p l a s m . M a g n i f i c a t i o n χ 15,700.
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Figure 7. Higher magnification of generative hyphal walls from N-limited nutrient agar grown cultures labeled for lignin-peroxidase. Double layer of label on both margins of the hyphal wall. Magnification χ 11,000.
Figure 8. Higher magnification of generative hyphal walls from N-limited nutrient agar grown cultures labeled for lignin-peroxidase. Label on the hyphal tip where a thickening of the wall and mucilage form a "cap" at the tip. Magnification χ 44,000.
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PLANT C E L L W A L L POLYMERS
w i t h v i r t u a l l y no l a b e l l i n g of the c y t o p l a s m , or of the mucilage s u r r o u n d i n g the w a l l . T h e difference i n p a t t e r n , a n d i n the degree of l a b e l l i n g of l i g n i n peroxidase i n sections of generative a n d skeletal h y p h a e are p r e s u m a b l y due t o the difference i n thickness of the w a l l . H y p h a e g r o w n o n m a l t agar showed s i m i l a r l a b e l l i n g of the p l a s m a m e m b r a n e w i t h l i g n i n - p e r o x i d a s e antisera, t h o u g h l a b e l l i n g of the cell w a l l was at a lower i n t e n s i t y t h a n t h a t seen i n hyphae c u l t u r e d o n N - l i m i t e d n u t r i e n t agar. Sections of decayed a n d undecayed beech h e a r t w o o d were also t r e a t e d w i t h l i g n i n - p e r o x i d a s e a n t i b o d i e s . F i g u r e 9 shows t h a t the l a b e l l i n g o f decayed w o o d was far more intense t h a n t h a t seen i n sections t r e a t e d w i t h laccase a n t i b o d i e s w i t h the l a b e l u n i f o r m l y d i s t r i b u t e d t h r o u g h o u t the S2 cell w a l l layer. L i t t l e l a b e l l i n g of the S I layer or m i d d l e l a m e l l a was o b served. O n sections of undecayed w o o d there was o n l y a v e r y slight a m o u n t o f b a c k g r o u n d l a b e l o n the cell walls i n d i c a t i n g t h a t there was l i t t l e n o n specific b i n d i n g to cell w a l l components, as seen i n F i g u r e 10. I n p r e l i m i n a r y e x p e r i m e n t s d i l u t i o n s of 1/100 of b o t h antisera were f o u n d t o give the clearest results w i t h m i n i m u m b a c k g r o u n d l a b e l l i n g yet s h o w i n g the highest i n t e n s i t y of specific l a b e l l i n g . L o w e r d i l u t i o n s gave less specific l a b e l l i n g , whereas higher d i l u t i o n s gave more non-specific l a b e l l i n g . C o n t r o l e x p e r i m e n t s to assess the specificity of the l a b e l l i n g were p e r f o r m e d by v a r i a t i o n s i n the procedure. F o r e x a m p l e , w h e n the p r i m a r y a n t i b o d y was o m i t t e d no l a b e l l i n g was observed, i n d i c a t i n g t h a t there was no non-specific b i n d i n g of goat a n t i - r a b b i t I g G - g o l d conjugate t o the sections. W h e n r a b b i t I g G i m m u n o g l o b u l i n was used to replace the p r i m a r y a n t i b o d y , again no l a b e l l i n g was seen i n d i c a t i n g t h a t non-specific b i n d i n g of i m m u n o g l o b u l i n s to sections was not responsible for l a b e l l i n g i n either h y p h a e or decayed w o o d cell w a l l s . S i m i l a r l y no l a b e l l i n g was observed w h e n the p r i m a r y a n t i b o d y was replaced w i t h antisera to p l a n t p h y t o c h r o m e ( F i g u r e 11). W h e n the p r i m a r y a n t i b o d y was replaced w i t h antisera to laccase i s o l a t e d f r o m Agaricus bisporus g r o w n i n m a l t e x t r a c t c u l t u r e s , the l a b e l l i n g p a t t e r n o b served was i d e n t i c a l to t h a t seen w i t h the p r i m a r y a n t i b o d y to laccase f r o m C. versicolor ( F i g . 12). T h i s suggests t h a t laccase f r o m A. bisporus has s i m i l a r antigenic sites to laccase f r o m C. versicolor. W h e n the p r i m a r y a n t i b o d y was replaced w i t h a n t i s e r a raised against the e n z y m e m a n n i t o l dehydrogenase, a c y t o p l a s m i c enzyme f r o m Agaricus bisporus, slight l a b e l l i n g was observed o n sections of the h y p h a e , associated w i t h b o t h the w a l l a n d c y t o p l a s m , a n observation w h i c h is difficult to e x p l a i n as m a n n i t o l dehydrogenase is a c y t o p l a s m i c enzyme i n A. bisporus, not e x t r a c e l l u l a r . W h e n sections of beech w o o d were treated w i t h a n t i s e r a to m a n n i t o l d e h y drogenase no l a b e l l i n g of the w o o d cell walls o c c u r r e d . T h i s i n d i c a t e d t h a t non-specific b i n d i n g was not t a k i n g place. Discussion T r a n s m i s s i o n electron m i c r o s c o p y of i m m u n o g o l d l a b e l l e d sections has s h o w n t h a t the e x t r a c e l l u l a r l i g n i n - d e g r a d i n g enzymes l i g n i n - p e r o x i d a s e a n d laccase were l o c a l i z e d w i t h i n the cell w a l l a n d mucilage of the h y p h a e of C. versicolor. Laccase was present i n the cell w a l l layer whereas l i g n i n -
31.
GALLAGHER ET A L
Lignocellubse-Degrading Enzymes
437
F i g u r e 9. Beech heartwood decayed for 12 weeks w i t h C. versicolor l a belled for lignin-peroxidase. T h e label is d i s t r i b u t e d u n i f o r m l y over the secondary w a l l w h i c h shows characteristic t h i n n i n g a n d m a r k e d r e d u c t i o n i n electron density. N o label occurs i n the m i d d l e l a m e l l a a n d cell corners. M a g n i f i c a t i o n χ 22,000.
438
PLANT C E L L W A L L
POLYMERS
F i g u r e 10. Undecayed beech h e a r t w o o d labelled for lignin-peroxidase, w i t h very l i t t l e label o n the secondary w a l l or m i d d l e l a m e l l a . M a g n i f i c a t i o n χ 9,800.
31.
GALLAGHER ET A L
Lignocellubse-Degrading Enzymes
439
F i g u r e 11. A single h y p h a f r o m an agar grown c u l t u r e of C. versicolor s h o w i n g an absence of l a b e l after r e p l a c i n g p r i m a r y antisera w i t h antisera to p l a n t p h y t o c h r o m e . M a g n i f i c a t i o n χ 17,400.
440
PLANT CELL WALL POLYMERS
Figure 12. A single hypha from an agar grown culture of C. versicolor showing labeling when the primary antisera was replaced with antisera to laccase from malt agar cultures of Agaricus bisporus. Magnification χ 37,800. All bars, 1 μχη. All tissue was unstained and consequently of low contrast.
31.
GALLAGHER ET A L
Lignocellubse-Degrading Enzymes
441
peroxidase was v i s u a l i s e d as a double layer o n the cell w a l l a n d adjacent to the p l a s m a m e m b r a n e . B o t h enzymes were l o c a l i z e d w i t h i n the S2 layer o f the secondary w a l l of beech h e a r t w o o d infected w i t h C. versicolor. Pene t r a t i o n o f the cell walls of w o o d was more pronounced for l i g n i n - p e r o x i d a s e t h a n for laccase b u t neither enzyme h a d penetrated to the m i d d l e l a m e l l a or cell corners. T h e w o o d sections were s i g n i f i c a n t l y degraded a n d i t is not k n o w n whether the diffusion o f enzymes i n t o the secondary w a l l o c c u r r e d after w i d e s p r e a d d e g r a d a t i o n of the lignocellulose or preceded secondary w a l l t h i n n i n g . O t h e r workers (23,24) have f o u n d t h a t i n P. chrysosponum, l i g n i n - p e r o x i d a s e was present as a c y t o p l a s m i c enzyme associated m a i n l y w i t h the p l a s m a m e m b r a n e i n h y p h a e g r o w n i n sawdust a n d l i q u i d c u l t u r e . G a r c i a et al. (23) d i d not find the enzyme localised w i t h i n the w o o d cell w a l l , whereas S r e b o t n i k et al. (24) showed ligninase was detected e x t r a c e l l u l a r l y after fixation i n p i c r i c a c i d . It is k n o w n t h a t secretion of l i g n i n peroxidase o n l y occurs d u r i n g the secondary m e t a b o l i c phase of g r o w t h i n l i q u i d cultures (25) a n d i t is probable t h a t t h i s also occurs i n w o o d . In our e x p e r i m e n t s , beechwood infected by C. versicolor showed decay p a t t e r n s t y p i c a l of advanced stages o f f u n g a l infection w h i c h w o u l d i m p l y t h a t the secondary m e t a b o l i c phase h a d been reached. F u r t h e r studies are required on the continuous d e g r a d a t i o n patterns r e s u l t i n g f r o m f u n g a l infections of w o o d to e x p l a i n a l l these results. T h e results of the c o n t r o l e x p e r i m e n t s w i t h antisera to f u n g a l proteins have led us to question the specificity of the antibodies to laccase a n d l i g n i n peroxidase. These enzymes are glycoproteins a n d b o t h c a r b o h y d r a t e a n d p r o t e i n moieties w i l l provide antigenic sites for the p o l y c l o n a l a n t i b o d i e s . However, u s i n g W e s t e r n b l o t t i n g techniques, the laccase a n t i s e r a d i d not cross-react w i t h l i g n i n - p e r o x i d a s e , nor d i d the l i g n i n - p e r o x i d a s e a n t i s e r a cross-react w i t h laccase. It is likely t h a t as e x t r a c e l l u l a r f u n g a l proteins are secreted t h r o u g h the mucilage-polysaccharide layer a r o u n d the h y p h a e the c a r b o h y d r a t e moieties of these glycoproteins have c a r b o h y d r a t e s t r u c t u r e s i n c o m m o n w i t h the h y p h a l w a l l layer. T h i s m a y e x p l a i n w h y a l l the a n t i b o d i e s to f u n g a l proteins w h i c h were tested p r o d u c e d l a b e l l i n g p a t t e r n s i n the w a l l , as some of the antigens w o u l d b i n d to c a r b o h y d r a t e c h a r a c t e r istic of f u n g a l cell walls. Unless deglycosylated proteins are used to raise a n t i b o d i e s the l a b e l l i n g patterns o b t a i n e d do not i n d i c a t e specifically the l o c a t i o n of the p r o t e i n moiety. However, this s t u d y has s h o w n t h a t a l l the f u n g a l tissues tested have a c o m m o n antigen w h i c h is not s h a r e d by the p l a n t a n d a n i m a l tissues tested. In order to u n d e r s t a n d the l a b e l l i n g patterns of laccase a n d lignin-peroxidase more fully, i t w i l l be necessary to s t u d y the apoenzymes separated f r o m c a r b o h y d r a t e before conclusions c a n be d r a w n about their d i s t r i b u t i o n i n hyphae d u r i n g p r i m a r y a n d secondary g r o w t h phases i n n a t u r a l substrates. A cknowledgment s W e t h a n k the f o l l o w i n g for gifts of antisera: D r . Β. Z . C h o w d h r y for I g G g l o b u l i n , D r . J . B . W . H a m m o n d for m a n n i t o l dehydrogenase a n t i s e r a , D r . B . T h o m a s for p l a n t p h y t o c h r o m e a n t i s e r a . W e t h a n k S E R C ( U K ) for the award of a p o s t g r a d u a t e s t u d e n t s h i p .
442
PLANT CELL WALL POLYMERS
Literature Cited 1. Tien, M.; Kirk, T . K. Science 1983, 221, 7-12. 2. Kuwahara, M.; Glenn, J . K.; Morgan, Μ. Α.; Gold, M . H. FEBS Letts. 1984, 169, 247)250. 3. Dodson, P. J.; Evans, C. S.; Harvey, P. J.; Palmer, J . M. FEMS Micro. Letts. 1987, 42, 17-22. 4. Schoemaker, Η. E.; Harvey, P. J.; Bowen, R. M.; Palmer, J . M . FEBS Letts. 1985, 183, 7-12. 5. Kersten, P. J.; Tien, M.; Kalyanaran, B.; Kirk, T . K. J. Biol. Chem. 1985, 260, 2609-2612. 6. Bollag, J. M.; Leonowicz, A. Appl.Environ. Micro. 1984, 48, 647-653. 7. Evans, C.S. FEMS Micro Letts. 1985, 27, 339-343. 8. Kawai, S.; Umezawa, T.; Higuchi, T. FEBS Letts. 1987, 210, 61-65. 9. Blanchette, R.A. Can. J. Bot. 1980, 1496-1500. 10. Blanchette, R. A. Appl. Environ. Micro. 1984, 48, 647-653. 11. Otjen, L.; Blanchette, R. Appl. Environ. Micro. 1985, 50, 568-572. 12. Messner, K.; Stachelberger, H. Trans. Brit. Mycol. Soc. 1984, 83, 209216. 13. Messner, K.; Foisner, R.; Stachelberger, H.; Rohr, M. Trans. Brit. My col. Soc. 1985, 84, 457-466. 14. Harvey, P. J.; Schoemaker, H. E.; Palmer, J . M . FEBS Letts. 1986, 195, 242-246. 15. Montgomery, R. A. P. In Decomposer Basidiomycetes; Frankland; Hedger; Swift, Eds.; Cambridge Univ. Press: 1982; 3, 51-65. 16. Horisberger, M . In Scanning Electron Microscopy; Johari, O., Ed.; 1981, 2, 9-13. 17. Kirk, T . K.; Schultz, E . ; Connors, W. J.; Lorenz, L.F.; Zeikus, J . G . Arch. Micro. 1978, 117, 277-285. 18. Bergman, B.; Lindblad, P.; Pettersson, Α.; Renstrom, E.; Tiberg, E . Planta 1985, 166, 329-332. 19. Reynolds, E . S. J. Cell Biol. 1968, 17, 208-212. 20. Fahraeus, G.; Reinhammer, B. Acta Chem. Scand. 1967, 21, 2367-2378. 21. Ouchterlony, O. Acta Path. Microbiol. Scand. 1949, 26, 507-515. 22. Westermark, U.; Eriksson, Κ. E . Acta Chem. Scand. 1974, B28, 204208. 23. Garcia, S.; Latge, J . P.; Prevost, M . C.; Leisola, M . Appl. Environ. Micro. 1987, 53, 2384-2387. 24. Srebotnik, E . ; Messner, K.; Foisner, R.; Pettersson, B. Curr. Micro. 1988, 16, 221-227. 25. Keyser, P.; Kirk, T . K.; Zeikus, J . G. J. Bact. 1978, 135, 790-797. RECEIVED May 19, 1989
