Chapter 10
An Improved Radiotracer Method for Studying Formation and Structure of Lignin Noritsugu Terashima Faculty of Agriculture, Nagoya University, Nagoya 464-01, Japan
Since it is impossible to isolate lignin in its unaltered state, or to depolymerize it quantitatively into known structural entities, it has been difficult to determine lignin structure in the cell wall directly. Among attempts to circumvent these difficulties, the improved radiotracer method has provided useful information unobtainable by other methods. In this approach, specific dual-labeling of structural units in protolignin in intact plant tissue was achieved by administration of H and C labeled lignin precursors to differentiating tree xylem. Subsequent analysis of the resulting tissue (or lignin isolated from the tissue) was then carried out. These double-labeling experiments provided quantitative information on the structure of protolignin, as well as the changes occurring during its removal. Extension of this improved technique to the dehydrogenative polymerization of monolignols in vitro provided a method of more closely simulating lignin biogenesis in the cell wall. 3
14
M a n y approaches have been employed t o a t t e m p t t o elucidate the s t r u c t u r e of l i g n i n . However, most studies dealt w i t h isolated l i g n i n s a n d u t i l i z e d a n u m b e r o f different p h y s i c a l a n d c h e m i c a l m e t h o d s . These have i n c l u d e d ^ - N M R , C - N M R , U V a n d I R spectroscopy, a n d the analysis o f d e g r a d a t i o n p r o d u c t s f r o m acidolysis, t h i o a c i d o l y s i s , alkaline nitrobenzene o x i d a t i o n , p e r m a n g a n a t e o x i d a t i o n , hydrogenolysis, a n d p y r o l y s i s . W h i l e these m e t h o d s p r o v i d e d i m p o r t a n t basic i n f o r m a t i o n o n t h e s t r u c t u r e o f isolated l i g n i n s , i t s s t r u c t u r e w i t h i n the cell w a l l s t i l l r e m a i n s a n open q u e s t i o n . T h i s is because (i) i n i t s n a t i v e state, l i g n i n is heterogeneous w i t h respect to i t s m a c r o m o l e c u l a r s t r u c t u r e , m o r p h o l o g i c a l l o c a t i o n a n d a s s o c i a t i o n w i t h c a r b o h y d r a t e s ; t h i s i n f o r m a t i o n is lost d u r i n g i t s i s o l a t i o n f r o m t h e 1 3
0097-6156/89/0399-0148$06.00/0 © 1989 American Chemical Society
10.
Formation & Structure of Lignin
TERÀSHIMA
149
cell w a l l ; a n d (ii) i t is i m p o s s i b l e to o b t a i n a l i g n i n s a m p l e w h i c h can be u n a m b i g u o u s l y considered to represent whole p r o t o l i g n i n ; moreover, ( i i i ) i t is v e r y difficult to d e p o l y m e r i z e l i g n i n q u a n t i t a t i v e l y i n t o k n o w n m o n o m e r i c or o l i g o m e r i c b u i l d i n g u n i t s b y established d e g r a d a t i v e m e t h o d s . In a t t e m p t s to c i r c u m v e n t these difficulties, p r o t o l i g n i n w i t h i n cell walls has been e x a m i n e d by means of n o n - d e g r a d a t i v e m e t h o d s such as U V , I R , R a m a n a n d N M R spectroscopy, S E M - E D X A a n d h i s t o c h e m i c a l a n a l y s i s . I n a d d i t i o n to these techniques, a d m i n i s t r a t i o n of r a d i o - l a b e l e d l i g n i n precursors to a c t i v e l y l i g n i f y i n g p l a n t tissue in vivo, followed b y a p p r o p r i a t e analyses of the l i g n i n , has c o n t r i b u t e d g r e a t l y to our c u r r e n t u n d e r s t a n d i n g of l i g n i n s t r u c t u r e . T h e specific l a b e l i n g of l i g n i n i n p l a n t tissue is u s u a l l y achieved b y a d m i n i s t r a t i o n of a n a p p r o p r i a t e precursor w h e n l i g n i f i c a t i o n is a c t i v e l y o c c u r r i n g . S u i t a b l e precursors i n c l u d e L - p h e n y l a l a n i n e , p - c o u m a r i c a c i d , ferulic a c i d , s i n a p i c a c i d , p - g l u c o c o u m a r y l a l c o h o l , c o n i f e r i n a n d s y r i n g i n . F o r g r a m i n a c e o u s p l a n t s , L - t y r o s i n e is also effective. L a b e l l e d precursors can be prepared b y replacement of either a specific h y d r o g e n or c a r b o n of the precursor w i t h H or C respectively. O f these precursors, the m o n o l i g n o l glucosides, w h i c h o c c u r n a t u r a l l y i n the c a m b i a l sap of g y m n o s p e r m s (1) a n d some angiosperms (2), have been s h o w n to be the most s u i t a b l e precursors for specific l a b e l i n g of l i g n i n i n m a n y plants (3,4). F o r e x a m p l e , m o n o l i g n o l glucosides were efficient precursors of l i g n i n i n b o t h p o p l a r (5) a n d rice plants (6), even t h o u g h these glucosides were not detected i n the l i g n i f y i n g tissues of these p l a n t s . F u r t h e r , since c i n n a m y l a l c o h o l - g l u c o s y l transferases are w i d e l y d i s t r i b u t e d i n the p l a n t k i n g d o m (7,8), t h i s s u g gests t h a t these glucosides m a y f u n c t i o n as u n i v e r s a l precursors for l i g n i n biosynthesis. 3
1 4
I n t h i s c h a p t e r , i m p r o v e m e n t s i n the a p p l i c a t i o n of the r a d i o t r a c e r m e t h o d s are discussed, u s i n g as a n e x a m p l e , the in vivo f o r m a t i o n of " c o n densed" s u b s t r u c t u r e s i n the l i g n i n m a c r o m o l e c u l e . A d d i t i o n a l l y , (i) the s t r u c t u r a l changes t h a t the l i g n i n m a c r o m o l e c u l e undergoes d u r i n g its rem o v a l b y c h e m i c a l means; (ii) the use of labeled s y n t h e t i c l i g n i n p r e p a r a t i o n s ; a n d (iii) the i m p o r t a n c e of c a r b o h y d r a t e s i n l i g n i f i c a t i o n , are d i s cussed. Materials and
Methods
Selective Labeling of a Specific Structural Unit in Protolignin. In the exp e r i m e n t s described, two to five year o l d shoots of a p p r o x i m a t e l y the same size were cut f r o m trees g r o w n under s i m i l a r c o n d i t i o n s . T o each s h o o t , the labeled precursor was a d m i n i s t e r e d t h r o u g h the cut e n d . A f t e r a predeterm i n e d p e r i o d for u p t a k e a n d m e t a b o l i s m , the b a r k tissue was r e m o v e d . T h e n e w l y formed x y l e m tissue c o n t a i n i n g cell walls at the same stage of differe n t i a t i o n was collected m e c h a n i c a l l y by c u t t i n g 100 μ M t a n g e n t i a l sections f r o m the c a m b i u m t o w a r d the inner p a r t of the x y l e m tissue by means of a sliding microtome. F o r radioassays, sections were m i l l e d to about 40 m e s h , a n d t h e n ex t r a c t e d successively w i t h e t h a n o l , benzene-ethanol a n d hot water. The
150
PLANT C E L L W A L L
POLYMERS
r e s u l t i n g tissue was t h e n subjected to c o m b u s t i o n to give, d e p e n d i n g u p o n the precursor a d m i n i s t e r e d , either H 2 0 , C C > 2 , or b o t h . R a d i o a c t i v i t y contents were t h e n d e t e r m i n e d b y l i q u i d s c i n t i l l a t i o n c o u n t i n g . T h e s t a n d a r d d e v i a t i o n of the assay was 2 % . 3
14
Synthesis of DHP (Dehydrogenative Polymer) from Coniferyl Alcohol-[ H, C] in the Presence of Carbohydrates. C o n i f e r y l alcohol-[ring 5 H , U - C ] (10 m g , H , 8300 d p m ; C , 3630 d p m ) was dissolved i n B r i t t o n R o b i n s o n buffer (2.0 m l ) c o n t a i n i n g the a p p r o p r i a t e p o l y s a c c h a r i d e (30 mg) a n d p e r oxidase (10 / i g , H o r s e r a d i s h T y p e II, S i g m a C o . , U S A ) to afford a g e l . H y drogen peroxide (0.5 m L , 0.5%) was slowly a d d e d by p e r m e a t i o n t h r o u g h a cellulose d i a l y s i s m e m b r a n e , spread over the end of a glass t u b e ( d i a m e t e r : 1.5 cm) a n d i n s e r t e d j u s t below the surface of the g e l . F o l l o w i n g p o l y m e r i z a t i o n at 25° C for 5 3 h , the water was removed s l o w l y i n a desiccator under s l i g h t l y reduced pressure. T h e r e a c t i o n m i x t u r e was t h e n m a c e r a t e d i n e t h a n o l (0.5 m L x 3 ) , to dissolve the soluble D H P f r a c t i o n a n d any low m o l e c u l a r weight entities, thereby l e a v i n g a n i n s o l u b l e l i g n i n - c a r b o h y d r a t e c o m p l e x ( L C C ) . T h e D H P p o l y m e r was t h e n o b t a i n e d b y c o m b i n i n g the e t h a n o l f r a c t i o n s , r e m o v i n g the solvent under reduced pressure, a n d t h e n r e d i s s o l v i n g the residue i n C ^ C b - E t O H (0.5 m L , 2:1 v / v ) . T h i s s o l u t i o n was t h e n p o u r e d i n t o d r y ether (10 m L ) , f o l l o w i n g w h i c h the p r e c i p i t a t e d D H P was collected b y c e n t r i f u g a t i o n ( 8 0 0 0 x g, 10 m i n ) . 3
3
14
3
1 4
1 4
Results and Discussion Specific Labeling of Protolignin. T h e m a j o r p a r t of softwood l i g n i n is c o n s t i t u t e d of g u a i a c y l l i g n i n . C o n s e q u e n t l y , ferulic a c i d is efficiently a n d i n t a c t l y i n c o r p o r a t e d i n t o g u a i a c y l l i g n i n i n pine (9). However, w h e n h a r d w o o d s are a d m i n i s t e r e d r a d i o l a b e l e d ferulic or s i n a p i c acids i n the l i g h t , c o n s i d erable m e t h o x y l a t i o n or d e m e t h o x y l a t i o n of g u a i a c y l or s y r i n g y l residues c a n o c c u r , t h e r e b y i n t e r c o n v e r t i n g s a i d precursors. T o some extent, such interconversions can be reduced by a d m i n i s t e r i n g these precursors i n the d a r k , e.g., w h e n [ r i n g - 2 - H ] ferulic or s i n a p i c acids were i n d i v i d u a l l y a d m i n i s t e r e d to p o p l a r shoots, the d i s t r i b u t i o n of l a b e l i n t o g u a i a c y l a n d s y r i n g y l c o m p o n e n t s was 80:20 a n d 27:73, respectively (10). T h i s d i s t r i b u t i o n was established by s u b j e c t i n g the l i g n i n to n i t r o b e n z e n e o x i d a t i o n , and d e t e r m i n i n g the r a d i o a c t i v i t y of the l i b e r a t e d aldehydes, v a n i l l i n a n d syringaldehyde. These precursor s c r a m b l i n g p r o b l e m s were essentially overcome b y a d m i n i s t e r i n g labeled coniferin a n d s y r i n g i n to Magnolia kobus D C (3). T h e l i g n i n so o b t a i n e d was then s u b j e c t e d to o x i d a t i o n as before, where i t was f o u n d t h a t 90 a n d 9 9 % of the a c t i v i t y of t o t a l aldehydes was present i n v a n i l l i n a n d s y r i n g a l d e h y d e , respectively (3), i.e., the glucosides were i n c o r p o r a t e d selectively into the l i g n i n p o l y m e r . [Note t h a t the m o n o l i g n o l s themselves are not n o r m a l l y used i n l a b e l i n g e x p e r i m e n t s , even t h o u g h they are the i m m e d i a t e precursors of l i g n i n . T h i s is because they can be p o l y m e r i z e d w i t h o u t a n y b i o c h e m i c a l c o n t r o l , as soon as they are i n contact w i t h tissue c o n t a i n i n g peroxidase a n d hydrogen peroxide, a n d c o u l d t h u s p o t e n t i a l l y give erroneous results.] 3
10.
TERASHIMA
151
Formation & Structure of Lignin
Analysis of the Structure and Reactions of Lignin by the Double-Labeling Technique. T a b l e I shows the t y p e of i n f o r m a t i o n t h a t can be o b t a i n e d b y means of d o u b l e - r a d i o l a b e l i n g e x p e r i m e n t s , u s i n g as a n e x a m p l e g y m n o s p e r m tissue. T h a t is, b y j u d i c i o u s use of d o u b l y - l a b e l e d precursors, i t is possible to a s c e r t a i n the extent of s u b s t i t u t i o n / c o n d e n s a t i o n reactions at selected a r o m a t i c r i n g p o s i t i o n s , as w e l l as d e m e t h y l a t i o n a n d ( p r o p a n o i d ) s i d e - c h a i n e l i m i n a t i o n reactions. S u c h strategies are described i n greater d e t a i l below, u s i n g b o t h g y m n o s p e r m s a n d angiosperms. T a b l e I. Use of D o u b l y labeled Biosynthesis Studies
Guaiacyl Lignin
Precursors in
Lignin
P o s i t i o n of L a b e l i n P r e c u r s o r Hi-
Information Attainable
A r o m . ring 5 A r o m . ring 2 A r o m . ring 6
A r o m . ring A r o m . ring A r o m . ring
Degree of s u b s t i t u t i o n at the p o s i t i o n o f a r o m a t i c r i n g labeled w i t h H
Methoxyl
A r o m . ring
Demethylation, demethoxylation
A r o m . ring 2 A r o m . ring 5 A r o m . ring 6
Side-chain C S i d e - c h a i n Cp Side-chain C
3
H
3
E l i m i n a t i o n of s i d e - c h a i n c a r b o n ( C , C ^ and C ) and formation of β-l s t r u c t u r e
a
a
7
7
Degree of Substitution at the Aromatic Ring. T h i s m e t h o d is based o n the fact t h a t w h e n a l i g n i n precursor, t r i t i a t e d at a specific p o s i t i o n i n the a r o m a t i c r i n g , is i n c o r p o r a t e d into the l i g n i n p o l y m e r a n d t h e n undergoes a s u b s t i t u t i o n reaction at t h a t p o s i t i o n , the c o r r e s p o n d i n g t r i t i u m l a b e l is e l i m i n a t e d . F o r e x a m p l e , i f [arom. r i n g - 5 - H , U - C ] coniferyl a l c o h o l (I) is i n c o r p o r a t e d i n t o l i g n i n , the degree of f o r m a t i o n of s u b s t r u c t u r e s V - V I I ( F i g . 1) can be e s t i m a t e d f r o m the degree of s u b s t i t u t i o n ( D . S . % ) . T h i s is c a l c u l a t e d as follows: 3
^
M
3
H/
1 4
C r a t i o of m o n o l i g n o l — H / 3
3
H/
1 4
1 4
1 4
C r a t i o of p o l y m e r
„
Λ / Χ
C r a t i o of m o n o l i g n o l
However, since the frequency of a r o m a t i c 4 - 0 - 5 d i a r y l ether s u b s t r u c ture ( V ) i n l i g n i n is low (11-13), the D . S . % really provides a n e s t i m a t e of the a m o u n t of " c o n d e n s e d " structures ( V I , V I I ) . T h i s is i l l u s t r a t e d i n the f o l l o w i n g e x a m p l e : W h e n [ r i n g - 5 - H , U - C ] ferulic a c i d , a precursor of I, was a d m i n i s t e r e d to Japanese black pine (Pinus thunbergii P a r i . ) , an aver age D . S . value of 5 6 % was o b t a i n e d . However, t h i s value v a r i e d w i d e l y f r o m 3 0 - 8 0 % d e p e n d i n g u p o n the stage of cell w a l l f o r m a t i o n e x a m i n e d . ( T h e different x y l e m sections were o b t a i n e d by means of a m i c r o t o m e ) (14,15). These results can be e x p l a i n e d as follows: d u r i n g i n i t i a l l i g n i f i c a t i o n , l i g n i n is deposited i n the m i d d l e l a m e l l a a n d has a h i g h D . S . (75-80%), t h e r e b y es t a b l i s h i n g t h a t significant " c o n d e n s a t i o n " has occurred (14). O n the other 3
1 4
3
F i g u r e 1. R e m o v a l of H at p o s i t i o n 5 of the g u a i a c y l r i n g of c o n i f e r y l a l c o h o l (I) by f o r m a t i o n of r i n g s u b s t i t u t e d s t r u c t u r e s ( V , V I , V I I ) d u r i n g dehydrogenative polymerization.
10.
153
Formation & Structure of Lignin
TERASHIMA
h a n d , the l i g n i n deposited i n the secondary walls h a d a m u c h lower D . S . value (30-75%), i n d i c a t i n g t h a t t h i s reaction was not as prevalent i n t h a t tissue. O t h e r factors such as t e m p e r a t u r e (15), p l a n t h o r m o n e a d d i t i o n (16), light (17), g r a v i t y (14), a n d p H of the precursor s o l u t i o n (15), also have an effect o n the D . S . value. T h i s can best be i l l u s t r a t e d b y e x a m p l e s : (i) g r o w t h of P. thunbergii at r o o m t e m p e r a t u r e (25-30°C) gave a l i g n i n w i t h more condensed u n i t s (48-73%) t h a n t h a t o b t a i n e d at 10°C (33-57%) (11); (ii) w h e n [ r i n g - 5 - H , U - C ] ferulic a c i d was a d m i n i s t e r e d i n a s o l u t i o n c o n t a i n i n g the p l a n t h o r m o n e , a u x i n ( I A A , 1 0 " ~ M ) , the degree of c o n d e n s a t i o n increased (59-87%), whereas w i t h abscisic a c i d ( A B A , 1 0 " M ) i t was re duced (24-64%); (iii) when the precursor solutions were a d m i n i s t e r e d at p H 5.2 a n d 8.0, the D . S . was higher i n the former case (54-79%) t h a n i n the l a t t e r (54-71%). In a l l cases, higher a n d lower D . S . values were f o u n d for l i g n i n s i n tissue sections nearest to the c a m b i u m a n d i n the tissue f u r thest f r o m the c a m b i u m (1000 μτη) respectively, (iv) W h e n a pine shoot g r o w i n g at a n angle of 45° was a d m i n i s t e r e d [ r i n g - 5 - H , U - C ] ferulic a c i d , the H / C r a t i o was lower i n the underside tissue t h a n i n the u p p e r s i d e , as i n d i c a t e d i n the D . S . (%) values s h o w n i n T a b l e II (14,15). T h i s i n d i c a t e d t h a t the l i g n i n i n compression w o o d of g y m n o s p e r m s h a d more condensed g u a i a c y l u n i t s (at C-5) t h a n the l i g n i n present i n the u p p e r s i d e w o o d , w h i c h more closely resembled n o r m a l w o o d . N o t e , t h o u g h , t h a t the g y m n o s p e r m gingko showed a m u c h s m a l l e r response; however, t h i s result was i n agreement w i t h other analyses e s t a b l i s h i n g gingko to be a n excep t i o n to most g y m n o s p e r m s (18). In the case of angiosperms ( p o p l a r , locust a n d oleander), however, the s i t u a t i o n was very different t h a n t h a t of pine ( T a b l e II). N o significant differences i n D . S . (%) values were observed at p o s i t i o n C - 5 of the g u a i a c y l c o m p o n e n t of a n g i o s p e r m l i g n i n between the u p p e r a n d lower tissues. T h e D S values o b t a i n e d for C - 2 a n d C - 6 also p r o v i d e d valuable i n f o r m a t i o n : a n d these are s h o w n for b o t h g y m n o s p e r m s a n d angiosperms (16). A s can be seen f r o m T a b l e II, these values were low i n a l l cases ( < 3.9%) a n d no discernible differences between upper a n d lower tissues were observable. 3
1 4
6
6
3
3
1 4
1 4
T h u s , these results are i n agreement w i t h other studies (19,20), (e.g., b y degradative analysis) where i t was found t h a t (i) g y m n o s p e r m compression w o o d l i g n i n differs f r o m " n o r m a l w o o d " l i g n i n b y v i r t u e of its h i g h content of condensed u n i t s , a n d (ii) tension w o o d l i g n i n i n angiosperms c o n t a i n s o n l y s l i g h t l y more condensed u n i t s t h a n n o r m a l w o o d l i g n i n . Retention of Propanoic! Side-Chains During Lignin Formation. As noted p r e v i o u s l y f r o m T a b l e II, the degree of s u b s t i t u t i o n at C - 2 of the g u a i a c y l r i n g of l i g n i n was low (0-2%). T h i s finding can be used to deter m i n e the extent of side-chain e l i m i n a t i o n as a result of f o r m a t i o n of β-1 s t r u c t u r e s ( F i g . 1, S t r u c t u r e I V ) . In order to determine t h i s , l a b e l i n g of the a p p r o p r i a t e l i g n i n precursor w i t h H at C - 2 of the a r o m a t i c r i n g , a n d C i n the s i d e - c h a i n , was carried o u t . O b v i o u s l y , i f l i t t l e or no e l i m i n a t i o n of the s i d e - c h a i n took place d u r i n g its i n c o r p o r a t i o n i n t o the l i g n i n p o l y m e r , t h e n the H / C r a t i o w o u l d r e m a i n essentially u n c h a n g e d . O n the 3
1 4
3
1 4
154
PLANT C E L L W A L L P O L Y M E R S
T a b l e II. Degree of S u b s t i t u t i o n (%) of the G u a i a c y l R i n g of L i g n i n i n the U p p e r a n d Lower P o r t i o n s of V a r i o u s Tree Shoots G r o w n at a 45° A n g l e
Pine Gingko* Italian Poplar K a m a b u c h i Poplar** Locust Oleander-' c
6
b
0
d
e
f 9
Position 6
Upper Lower (DS % ) '
U p p e r Lower (DS %)»
52.1 49.9 49.9 54.1 46.2 47.9
a
a
Position 5
62.2 51.8 49.9 51.1 44.0 47.3
3.9 0.0 3.0 2.0 3.0 0.0
Pinus thunbergii P a r i . Ginkgo biloba L . Populus euramericana cv. '1-214'. Populus nigra L . X Populus maximiwizii Robinia pseudoacacia L . Nerium indicum M i l l . D S % = degree of s u b s t i t u t i o n (%).
3.9 0.0 3.0 2.0 3.0 0.0
Position 2 Upper (DS 2.0 0.0 1.9 2.0 1.9 0.0
Lower %y 2.0 0.0 0.0 2.0 1.9 0.0
A . Henry.
other h a n d , a n increase w o u l d signify the involvement of s i d e - c h a i n e l i m i n a t i o n . These l a b e l i n g studies showed, however, t h a t l i t t l e or no e l i m i n a t i o n o c c u r r e d (21). In agreement w i t h this finding, ^ - N M R e x a m i n a t i o n of m i l l e d w o o d l i g n i n f r o m spruce a n d b i r c h also revealed t h a t the β-l content was very low (22,23). Retention of Methoxyl Groups During the Formation of Lignin i n v i v o . F o l l o w i n g u p t a k e of [ r i n g - 2 - H , 0 C H 3 ] ferulic acid to g r o w i n g stems of pine a n d l o c u s t , a n d subsequent analysis of the r e s u l t i n g l i g n i n , it was f o u n d t h a t no significant d e m e t h y l a t i o n or d e m e t h o x y l a t i o n of the g u a i a c y l nucleus occurs d u r i n g l i g n i n f o r m a t i o n (15). 3
1 4
Changes to the Macromolecule during Delignification. A s discussed beforehand, there is no m e t h o d c u r r e n t l y available for the i s o l a t i o n of p r o t o l i g n i n i n its u n a l t e r e d state. O n the other h a n d , w h i l e there are m a n y techniques used to isolate l i g n i n , the effect of these c h e m i c a l t r e a t m e n t s o n the s t r u c t u r e of the m a c r o m o l e c u l e is p o o r l y u n d e r s t o o d . A s p r e v i ously m e n t i o n e d , since the e l i m i n a t i o n of side-chain a n d m e t h o x y l groups scarcely occurs d u r i n g g y m n o s p e r m l i g n i n f o r m a t i o n , the c a r b o n skeleton of g u a i a c y l - r i c h p r o t o l i g n i n can be considered to be m a i n l y C 6 - C 3 - O C H 3 . T h u s , specifically labeled g u a i a c y l l i g n i n can be used to determine some of the changes t h a t it undergoes d u r i n g various c h e m i c a l t r e a t m e n t s . T h e labeled g u a i a c y l l i g n i n used i n these studies was either present i n intact tissue of pine, or i n a n isolated m i l l e d w o o d l i g n i n p r e p a r a t i o n (24). T a b l e III s u m m a r i z e s the results o b t a i n e d . A s can be seen, the lignins isolated by solvolysis w i t h e t h a n o l , d i m e t h o x y p r o p a n e , b e n z y l e t h y l ether or dioxane h a d very s i m i l a r H / C ratios to that of the o r i g i n a l p r o t o l i g n i n (24). O n 3
1 4
10.
TERASHIMA
155
Formation & Structure of Lignin
the other h a n d , a n d as can be seen f r o m the H / C ratios o b t a i n e d , s u b s t a n t i a l s t r u c t u r a l changes o c c u r r e d d u r i n g the i s o l a t i o n of K l a s o n (24) a n d kraft (25) l i g n i n s , w i t h appreciable a m o u n t s of b o t h m e t h o x y groups a n d s i d e - c h a i n carbons b e i n g lost. F r o m these i n v e s t i g a t i o n s , i t can thus be c o n c l u d e d t h a t the average r e p e a t i n g u n i t i n kraft l i g n i n is C6-C2.32(OCH3)o.76> i.e., significant m o d i f i c a t i o n s to the c a r b o n skeleton have o c c u r r e d (25). E s t i m a t i o n b y C N M R gave s i m i l a r results for loss of s i d e - c h a i n c a r b o n s for pine k r a f t l i g n i n (26). 3
1 4
1 3
T a b l e I I I . R e l a t i v e N u m b e r s of M e t h o x y l a n d S i d e - C h a i n C a r b o n s to G u a i a c y l R i n g C a r b o n s i n V a r i o u s L i g n i n D e r i v a t i v e s Isolated f r o m Pinus thunbergii (25,26)
Lignin Type P r o t o l i g n i n i n cell w a l l B e n z y l e t h y l ether l i g n i n " Dimethoxypropane lignin Ethanolysis lignin Dioxane lignin Klason lignin Kraft lignin c
a
h
c
6
Arom. Ring
Methoxy
6.00 6.00 6.00 6.00 6.00 6.00 6.00
1.00 1.04 0.92 0.99 0.92 0.87 0.76
C
c
a
1.00 0.97 0.98 0.95 1.08 0.92 0.79
1.00 0.99 1.00 0.95 0.95 0.92 0.84
7
1.00 0.95 1.05 0.96 1.07 0.84 0.69
SideChain 3.00 2.91 3.03 2.86 3.10 2.68 2.32
S o l v o l y s i s l i g n i n prepared by t r a n s e t h e r i f i c a t i o n (24). S o l v o l y s i s l i g n i n prepared w i t h 2 , 2 - d i m e t h o x y p r o p a n e (24). W o o d m e a l s i n g l y labeled w i t h C was e m p l o y e d . T h i s e s t i m a t i o n was m a d e o n the basis of r a d i o a c t i v i t y a n d U V absorbance (25). 1 4
Dehydrogenative Polymerization of Monolignols i n v i t r o . A s observed f r o m the m i c r o a u t o r a d i o g r a m s of n e w l y formed x y l e m of pine (P. thunbergii), the early stages of cell w a l l development l i g n i n d e p o s i t i o n are a l w a y s preceded b y d e p o s i t i o n of pectic substances (27), a n d then by h e m i c e l l u loses i n the later stages (27). Interestingly, the l i g n i n m a c r o m o l e c u l e i n the c o m p o u n d m i d d l e l a m e l l a deposited d u r i n g these early stages of cell w a l l differentiation c o n t a i n s more "condensed" u n i t s t h a n t h a t formed later i n the secondary w a l l (14,15). T h i s c o u l d be due to the influence of several factors, such as (i) pectic substances a n d m a n n a n s affecting the process of dehydrogenative p o l y m e r i z a t i o n of the m o n o l i g n o l s (28); (ii) the c o n c e n t r a t i o n of peroxidase b e i n g higher i n the cell corner a n d m i d d l e l a m e l l a regions (29). T h i s c o u l d result i n the f o r m a t i o n of a more " c o n d e n s e d " l i g n i n (28); ( i i i ) p - h y d r o x y p h e n y l p r o p a n e u n i t s p a r t i c i p a t i n g more extensively d u r i n g the f o r m a t i o n of m i d d l e l a m e l l a l i g n i n (4,30). S u c h influences can be exa m i n e d , at least i n a crude way, by s y n t h e s i z i n g labeled d e h y d r o g e n a t i v e p o l y m e r ( D H P ) f r o m [ r i n g - & - H , U - C ] c o n i f e r y l a l c o h o l under c o n d i t i o n s a p p r o x i m a t i n g these s i t u a t i o n s . 3
1 4
Effect of C a r b o h y d r a t e : T a b l e I V shows the effect of c a r b o h y d r a t e s o n the D . S . (%) of b o t h l i g n i n - c a r b o h y d r a t e complexes ( L C C ' s ) a n d D H P ' s
156
PLANT C E L L W A L L POLYMERS
p r o d u c e d f r o m [ r i n g - 5 - H , U - C ] coniferyl a l c o h o l at p H 5.5, 6.5 a n d 7.5, respectively. A s can be seen, the D . S . ( % ) of D H P ' s f o r m e d i n the presence of x y l a n was greater t h a n t h a t observed for the D H P ' s f o r m e d i n the absence of polysaccharides. A s far as the L C C ' s were concerned, the D . S . (%) values w i t h x y l a n a n d p e c t i n were also higher t h a n t h a t for D H P ' s p r o d u c e d solely from coniferyl alcohol. 3
1 4
T a b l e I V . T h e Effect of C a r b o h y d r a t e s on the Degree of S u b s t i t u t i o n at P o s i t i o n 5 of G u a i a c y l R i n g i n D H P a n d L C C F r a c t i o n s
Carbohydrate
pH
None None None Xylan Xylan Xylan M an η a n Mannan Mannan Pectin Pectin Pectin Pectin
5.5 6.5 7.5 5.5 6.5 7.5 5.5 6.5 7.5 5.0 6.5 6.5d 7.5d
a
6
0
a
b
c
d
e
D.S.% of D H P
Yield (%)
D.S.% of L C C
Yield (%)
42.7 39.5 35.0 51.3 43.3 44.3 40.0 35.6 35.6
47.6 41.8 44.4 46.7 45.3 43.2 44.5 45.3 40.9
-
n.d.
45.2
22.9 21.0 18.0 18.0 15.6 19.9 n.d. 27.8 28.6 n.d.
-
-
41.4 37.9 26.2
29.5 37.4
-
45.5 35.1 28.0 25.5 36.4 45.1 41.8 35.1 36.6
e
-
Isolated f r o m c o t t o n seed hulls by d e l i g n i f i c a t i o n w i t h chlorous a c i d followed b y e x t r a c t i o n w i t h s o d i u m h y d r o x i d e . O b t a i n e d f r o m m a n u f a c t u r e r of k o n n y a k u , a type of f o o d m a d e f r o m the tuber of the konyak p l a n t (Amorphophallus konjac C . K o c h ) . C i t r u s p e c t i n purchased f r o m T o k y o K a s e i C o . , T o k y o . 3 m g of c a l c i u m h y d r o x i d e was a d d e d . n . d . = not d e t e r m i n e d .
Effect of p H : T h e effect of p H was also i n t e r e s t i n g . A s can be seen ( T a ble I V ) , the D . S . ( % ) values for the p o l y m e r s formed at low ρ Η were higher t h a n those at h i g h p H . N e x t we e x a m i n e d the effect of c a l c i u m c a t i o n s , since c a l c i u m is supposed to p a r t i c i p a t e i n the l i g n i f i c a t i o n process (31). D i r e c t a d d i t i o n of c a l c i u m h y d r o x i d e to the reaction m i x t u r e lowered the D . S , b u t this was p r o b a b l y o n l y a consequence of increased p H . T h e effects noted for p e c t i n a n d x y l a n can be p a r t l y ascribed to t h e i r acidic p r o p e r t i e s . These results m a y therefore e x p l a i n the observation t h a t when a pine shoot is a d m i n i s t e r e d [ r i n g - 5 - H , r i n g - U - C ] ferulic a c i d , dissolved i n p h o s p h a t e buffer at p H 5.2 a n d 8.0, the D . S . ( % ) value due to c o n d e n s a t i o n at C - 5 of the l i g n i n was higher i n the shoot a d m i n i s t e r e d at low p H (15). T h i s p H ef fect can p o t e n t i a l l y be e x p l a i n e d as follows: F r e u d e n b e r g et ai established t h a t coniferyl a l c o h o l is first dehydrogenated by m u s h r o o m l a c c a s e / 0 2 or p e r o x i d a s e / H 2 0 2 to y i e l d the transient p h e n o x y r a d i c a l species, s h o w n i n 3
1 4
10.
TERASHIMA
157
Formation & Structure of Lignin
F i g u r e 1. A m o n g the five resonance structures s h o w n , R a , R b a n d R c w i l l undergo r a n d o m c o u p l i n g reactions more r e a d i l y t h a n R d a n d R e , since the r a d i c a l s R d a n d R e are s t e r i c a l l y h i n d e r e d . Indeed, this is w h a t is f o u n d e x p e r i m e n t a l l y , since o n l y m i n o r a m o u n t s of β A s t r u c t u r e s (derived f r o m R d ) have been f o u n d i n m i l l e d w o o d l i g n i n (22,23), a n d d e m e t h o x y l a t i o n does not occur to any appreciable extent d u r i n g l i g n i n f o r m a t i o n (21). T h e r e a c t i v i t y of the p h e n o x y r a d i c a l , R a , w i l l be affected g r e a t l y b y p H since it can be masked b y protons under a c i d i c c o n d i t i o n s , a n d its r e a c t i v i t y w i l l thus be d i m i n i s h e d . O n the other h a n d , the r e a c t i v i t y of R b a n d R c w i l l not be affected a p p r e c i a b l y b y p H . T h u s more frequent c o u p l i n g of R b - R c , R b - R b a n d R c - R c can be expected at low p H , a n d more of R a - R b a n d R a - R c c o u p l i n g at h i g h p H . For some t i m e n o w , it has been proposed t h a t the s t r u c t u r a l v a r i a t i o n s observed i n D H P ' s are caused by the mode of p o l y m e r i z a t i o n , w i t h b u l k p o l y m e r i z a t i o n c o n t a i n i n g more condensed u n i t s t h a n end-wise p o l y m e r i z a t i o n (32,33). However, i n these e x p e r i m e n t s , D H P ' s prepared under the same c o n d i t i o n s gave different D . S . values d e p e n d i n g u p o n the p H at w h i c h the r e a c t i o n took place. T h i s result suggests t h a t the m o l e c u l a r s t r u c t u r e of l i g n i n i n the cell w a l l can also be controlled by factors other t h a n the m o d e of p o l y m e r i z a t i o n . Condensed Structures in p-Hydroxyphenyl-Guaiacyl Type DHP. It has been difficult to determine the exact a m o u n t of p - h y d r o x y p h e n y l p r o p a n e u n i t s i n l i g n i n . T h i s can best be i l l u s t r a t e d by a n e x a m p l e ; n i t r o b e n zene o x i d a t i o n of a D H P p r e p a r e d b y the Z u t r o p f v e r f a h r e n m e t h o d f r o m a m i x t u r e of p - c o u m a r y l a l c o h o l , coniferyl a l c o h o l a n d s i n a p y l a l c o h o l , gave no detectable p - h y d r o x y b e n z a l d e h y d e o n alkaline nitrobenzene o x i d a t i o n (34). T o d e t e r m i n e the reasons for this, D H P ' s were prepared f r o m a m i x ture of [ r i n g - 2 - H ] p - c o u m a r y l alcohol a n d [ a - C ] c o n i f e r y l a l c o h o l i n the presence of c a r b o h y d r a t e s by the procedure described above (35). The " L C C " f r a c t i o n so o b t a i n e d was subjected to c o m b u s t i o n , a n d the exact p - h y d r o x y p h e n y l p r o p a n e : g u a i a c y l p r o p a n e r a t i o was d e t e r m i n e d f r o m the a c t i v i t y of H 0 a n d C 0 released ( F i g u r e 2). A p o r t i o n of the same L C C f r a c t i o n was also o x i d i z e d w i t h nitrobenzene a n d a l k a l i , a n d the re s u l t i n g l i b e r a t e d a r o m a t i c aldehydes were then a n a l y z e d b y H P L C . R e s u l t s are s h o w n i n T a b l e V . 3
3
2
1 4
1 4
2
T a b l e V . M o l a r R a t i o s of p - H y d r o x y p h e n y l ( H ) to G u a i a c y l ( G ) U n i t s i n D H P ' s a n d their N i t r o b e n z e n e O x i d a t i o n P r o d u c t s
Starting monolignol mixtures" " L C C " fraction of polymers" O x i d a t i o n products* α
b
H:G
H:G
H:G
2.00:1.00 1.88:1.00 1.21:1.00
1.00:1.00 0.90:1.00 0.72:1.00
0.50:1.00 0.47:1.00 0.41:1.00
E s t i m a t e d f r o m the r a d i o a c t i v i t i e s of H 2 Û a n d C0 p r o d u c e d by combustion. M o l a r ratios of p - h y d r o x y b e n z a l d e h y d e to v a n i l l i n were d e t e r m i n e d b y HPLC. 3
1 4
2
158
PLANT CELL WALL POLYMERS
CH OH 2
^
H
H
C
Combustion
DHP C6H5NO2 NaOH Figure 2. Dehydrogenative polymerization of a mixture of p-coumaryl alcohol-[ring-2- H] and coniferyl alcohol-[U- C], and nitrobenzene oxidation of the DHP to give p-hydroxybenzaldehyde-[ring-2- H] and vanillin[formyl- C]. 3
14
3
14
As can be seen, the molar ratios of p-hydroxyphenyl to guaiacyl units were slightly lower for the DHP's when compared to the original mixtures. This implies that coniferyl alcohol tends to be incorporated into the polymer slightly more readily than p-coumaryl alcohol. On the other hand, the much-reduced ratio of p-hydroxybenzaldehyde to vanillin, liberated during alkaline nitrobenzene oxidation, proved that this DHP contained a larger amount of condensed p-hydroxyphenylpropane units than condensed guaiacyl units. Finally, it should be noted that the structure of the DHP varies greatly depending on the polymerization conditions employed. Additionally, the yield and chemical and physical properties of these DHP preparations differ substantially from protolignin. Further improvements in simulation of the lignification process are therefore needed, and the radiotracer method can be employed as one approach to solve such problems. Concluding Remarks 1. The specific labeling of specific moieties in protolignin can be achieved by administration of an appropriate labeled precursor to a growing plant. 2. Double labeling with H and C at specific positions of an appropriate structural moiety in lignin, combined with accurate determination of H / C ratios, provides reliable information concerning protolignin structure and structural changes that occur during delignification. 3. The double labeling technique, combined with the technique of collection of xylem tissue at different stages of differentiation, provides additional information on the distribution of specific lignin substructures such as "condensed units" in different morphological regions. 4. The double-labeling technique is also useful for in vitro studies on the mechanism of dehydrogenative polymerization of monolignols. 5. This improved radiotracer method can be employed as a non-degradative method for examining the distribution, structure and reactions of lignin within the cell wall. 3
3
1 4
1 4
Literature Cited 1. Freudenberg, K.; Harkin, J. M . Phytochemistry 1963, 2, 322. 2. Terazawa, M.; Okuyama, H.; Miyake, M . Mokuzai Gakkaishi 1984, 30, 322.
10.
TERASHIMA
Formation & Structure of Lignin
159
3. Terashima, N.; Fukushima, K.; Takabe, K. Holzforschung 1986, 40, Suppl., 101. 5. Fukushima, K.; Terashjima, N. Proc. 32nd Lignin Symp. at Fukuoka 1987, p. 13. 6. He, L-F.; Terashima, N. Mokuzai Gakkaishi 1988, 35, 117. 7. Ibrahim, R. K.; Grisebach, H. Arch. Biochem. Biophys. 1976, 176, 100. 8. Ibrahim, R. Κ. Z. Pflanzenphysiol. 1977, 85, 253. 9. Terashima, N.; Suganuma, N.; Araki, H.; Kanda, T. Mokuzai Gakkaishi 1976, 22, 450. 10. Terashima, N.; Fukushima, K.; Tsuchiya, S.; Takabe, K. J. Wood Chem. Technol. 1986, 6, 495. 11. Erickson, M.; Larsson, S.; Miksche, G. E . Acta Chem. Scand. 1973, B27, 903. 12. Nimz, H. Angew. Chem. Int. Engl. Ed. 1974, 13, 313. 13. Glasser, W. G.; Glasser, H. R. Pap. Puu 1981, 63, 71. 14. Terashima, N.; Tomimura, Y.; Araki, H. Mokuzai Gakkaishi 1979, 25, 595. 15. Tomimura, Y.; Yokoi, T.; Terashima, N. Mokuzai Gakkaishi 1979, 25, 743. 16. Tomimura, Y.; Yokoi, T.; Terashima, N. Mokuzai Gakkaishi 1980, 26, 37. 17. Terashima, N. Proc. 32nd Ann. Mtg. Jap. Wood Res. Soc. 1982, p. 238. 18. Timell, T . E . Wood Sci. Technol. 1978, 12, 89. 19. Morohoshi, N.; Sakakibara, A. Mokuzai Gakkaishi 1971, 17, 393. 20. Yasuda, S.; Sakakibara, A. Mokuzai Gakkaishi 1975, 21, 363. 21. Tomimura, Y.; Terashima, N. Mokuzai Gakkaishi 1979, 25, 427. 22. Lundquist, K. Acta Chem. Scand. 1980, B34, 21. 23. Lundquist, K. Acta Chem. Scand. 1981, B35, 497. 24. Kachi, S.; Araki, H.; Terashima, N.; Kanda, T . Mokuzai Gakkaishi 1975, 21, 669. 25. Terashima, N.; Araki, H.; Suganuma, N. Mokuzai Gakkaishi 1977, 23, 343. 26. Robert, D. R.; Bardet, M.; Gellerstedt, G.; Lindfors, E . L. J. Wood Chem. Technol. 1984, 4, 239. 27. Terashima, N.; Fukushima, K.; Takabe, K. Holzforschung 1988, 42, 347. 28. Terashima, N.; Seguchi, Y. Cell. Chem. Technol. 1988, 22, 147. 29. Takabe, K.; Harada, H. Proc. 30th Lignin Symp. at Kochi 1985, p. 1. 30. Whiting, P.; Goring, D. A. I. Wood Sci. Technol. 1982, 16, 261. 31. Wardrop, A. B. Appl. Polym. Symp. 1976, 28, 1041. 32. Sarkanen, Κ. V. In Lignins: Occurrence, Formation, Structure and Re actions; Sarkanen, Κ. V.; Ludwig, C. H., Eds.; Wiley-Interscience: New York, 1971, p. 150. 33. Lai, Y-Z.; Sarkanen, Κ. V. Cell. Chem. Technol. 1975, 9, 239. 34. Faix, V. Ο.; Schweers, W. Holzforschung 1975, 29, 48. 35. Terashima, N.; Proc. 38th Ann. Mtg. Jap. Wood Res. Soc. 1988, p. 368. RECEIVED March 27, 1989
