Plant Cell Wall Polymers - American Chemical Society

190. PLANT CELL WALL POLYMERS peatedly suggested (24,38,39,40). Likely the ... ilar to values reported for the b.m.l-type by these authors (20,21). Th...
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Chapter 13

Biochemical and Biosynthetic Studies on Lignification of Gramineae Monique Gaudillere and Bernard Monties Laboratoire de Chimie Biologique, INRA, Institut National Agronomique Paris-Grignon, Centre de Grignon, 78850 Thiverval-Grignon, France

Differences in lignification of forage crops were exam­ ined in terms of genetic, biosynthetic and environmen­ tal factors. This was achieved by comparison of brit­ tle ecotypes of fescue (Festuca arundinacea), and brown­ -midrib (b.m.3)-mutants of maize (Zea mays) with the corresponding "normal" plants. For each plant type, Klason, acid-insoluble and acetyl bromide lignin con­ tents, and monomeric compositions, were determined and compared. While only weak differences in lignifi­ cation were found in the case of fescue, significant dif­ ferences in both lignin content, and its monomeric com­ position, were found for maize between its upper and lower internodes. These differences were due to genetic and/or environmental factors. Heterogeneity in lignifi­ cation, brittle organ character, and other biosynthetic aspects of stem formation in Gramineae are discussed, in relation to results previously obtained with rice and wheat. Forages such as hay a n d straws o f the G r a m i n e a e are p r o d u c e d for r u m i n a n t feed o n a n enormous scale. I n France, a n n u a l p r o d u c t i o n is 12.8 χ Ι Ο Τ (1) w h i c h approaches t h a t for w o o d - h a r v e s t i n g operations (23 χ 1 0 T ) (2). A s described i n preceding chapters, l i g n i n s (and related aromatics) appear t o b i n d p h y s i c a l l y a n d / o r c h e m i c a l l y t o the cell w a l l polysaccharides o f forages. These a r o m a t i c substances have a p r o f o u n d effect o n a n i m a l n u t r i t i o n ( 3 10). Differences between forage d i g e s t i b i l i t y have also been correlated w i t h e n v i r o n m e n t a l a n d n u t r i t i o n a l factors (11), as well as species a n d genetic v a r i a b i l i t i e s (12,13). A s regards l i g n i n (14) a n d c e l l - w a l l - b o u n d h y d r o x y c i n n a m i c acids (15) (i.e., p - c o u m a r i c , ferulic acids), i t is now generally accepted t h a t a n y increase i n their content results i n decreased d i g e s t i b i l i t y . R u m i ­ n a n t d i g e s t i b i l i t y o f p l a n t m a t e r i a l can be i m p r o v e d b y either m i l d a c i d (16) 6

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0097-6156/89/0399-0182$06.00/0 © 1989 American Chemical Society

13.

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Lignification of Gramineae

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or, more u s u a l l y , a l k a l i (17-18) t r e a t m e n t to remove labile phenolics a n d l i g n i n - c a r b o h y d r a t e f r a c t i o n s , thus l e a v i n g b e h i n d a more readily-accessible p o l y s a c c h a r i d e feed. Interestingly, differences i n d i g e s t i b i l i t y between c u l t i vars can be higher t h a n differences r e s u l t i n g f r o m c h e m i c a l t r e a t m e n t (19), t h u s u n d e r s c o r i n g the i m p o r t a n c e of b i o l o g i c a l v a r i a b i l i t y . T o date, most conclusions m a d e r e g a r d i n g d i g e s t i b i l i t y differences of m a i z e , s o r g h u m a n d rice, have been e x p l a i n e d o n the basis of v a r i a t i o n s i n l i g n i n contents. In the case of m a i z e (Zea mays L . ) , a f a m i l y of ten single- or d o u b l e - m u t a n t s was first observed by K u c et al. (20,21). S u c h m u t a n t s were d i s t i n g u i s h e d by a b r o w n i n g of the m i d - r i b s of the leaves ( b r o w n - m i d r i b : b . m - m u t a n t ) , b y a lower l i g n i n content a n d higher d i g e s t i b i l i t y . In these m u t a n t s , differences i n b o t h l i g n i n m o n o m e r c o m p o s i t i o n (following nitrobenzene o x i d a t i o n ) a n d i n the relative content of cell-wall-ester l i n k e d p - c o u m a r i c a n d ferulic acids were observed. B r o w n - m i d r i b m u t a n t s of s o r g h u m (Sorghum bicolor L . M o e n s c h ) were the second f a m i l y of m u t a n t s s t u d i e d , a n d were also c h a r acterized by a lower l i g n i n content (22-23). A m u t a n t of rice (Oriza sativa L . ) was also f o u n d , a n d was r e a d i l y d i s t i n g u i s h a b l e b y a brittleness of the c u l m w h i c h appeared o n l y after m a t u r i t y of the p l a n t . T h i s m u t a n t h a d a lower cellulose content, a n d t h i s difference was assumed to be related to the brittleness of the c u l m (24). Significant differences were also f o u n d i n the e x t r a c t a b i l i t y of the l i g n i n fractions a n d associated p h e n o l i c acids (25-26), suggesting t h a t l i g n i n f o r m a t i o n was also affected. In t h i s w o r k , our m a i n o b j e c t i v e was to explore the u n d e r l y i n g reasons for such v a r i a t i o n s i n g r a m i n a c e o u s l i g n i n s . A s before for rice (26), p h e n o l i c ester a n d l i g n i n analyses (content a n d m o n o m e r c o m p o s i t i o n ) were carried o u t , a l t h o u g h o n l y l i g n i n d a t a are s h o w n here. F i r s t l y , we s t u d i e d possible r e l a t i o n s h i p s between l i g n i n v a r i a t i o n a n d brittleness of p l a n t organs, u s i n g two ecotypes of t a l l fescue grass (Festuca arundinacea S c h r e b ) . T h u s , b o t h " n o r m a l " fescue a n d a b r i t t l e ecotype (discovered b y J a d a s - H e c a r t (27)), characterized by a brittleness of leaves, s h e a t h a n d s t e m , were c o m p a r e d . Possible e n v i r o n m e n t a l effects o n the b i o c h e m i s t r y of l i g n i n f o r m a t i o n were e s t i m a t e d by c o m p a r i s o n of several p a r a l l e l crops f r o m two l o c a t i o n s . Secondly, a b i o s y n t h e t i c i n v e s t i g a t i o n on l i g n i n v a r i a t i o n was u n d e r t a k e n u s i n g m a i z e internodes. M a i z e internodes were e x a m i n e d i n t h i s s t u d y since: (a) fewer plants were required for a n a l y s i s (greater b i o m a s s ) ; a n d (b) possible v a r i a t i o n s between n o r m a l a n d b . m - m u t a n t s (21) c o u l d be s t u d i e d . L i g n i n contents a n d m o n o m e r c o m p o s i t i o n were c o m p a r e d between internodes, b o t h being collected at the top a n d the b o t t o m of the m a i z e s t e m . These p l a n t p a r t s were chosen because of differences i n the d i g e s t i b i l i t y of different internodes as d o c u m e n t e d for T i m o t h y (Phleum pratense) (28) a n d , i n l i g n i f i c a t i o n , for wheat (Triticum aestivum L . ) (29-30). Materials and

Methods

Material. P l a n t s were g r o w n under field or greenhouse c o n d i t i o n s at G r i g n o n , a n d harvested before h e a d i n g for Fescue (27), a n d at g r a i n m a t u r i t y i n the case of m a i z e (31). In each case, p l a n t s were harvested at the

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PLANT C E L L W A L L P O L Y M E R S

same stage of m a t u r i t y , as s h o w n b y the development of inflorescence, a n d the r e l a t i v e r a t i o of leaves a n d s t a l k . Methods. W h o l e p l a n t s were harvested i n the case of fescue leaves, whereas for m a i z e s t a l k s , sheaths a n d nodes were removed, l e a v i n g internodes w h i c h were w h o l l y a n a l y z e d . S a m p l e s (6 to 10 plants) were freeze d r i e d , finely ground and exhaustively extracted, i n a Soxhlet, w i t h toluene-ethanol ( 2 / l : v / v ) , e t h a n o l , then w a t e r , l e a v i n g an i n s o l u b l e " p a r i e t a l residue" ( P R ) w h i c h was freeze d r i e d before storage a n d analysis. L i g n i n determ i n a t i o n s used three different m e t h o d s ; K l a s o n l i g n i n ( K L ) ( 7 2 % H 2 S O 4 ) ; a c i d - i n s o l u b l e l i g n i n ( A I L ) ( 5 % H 2 S O 4 p r e h y d r o l y s i s followed b y K l a s o n d e t e r m i n a t i o n ) ; a n d a c e t y l b r o m i d e l i g n i n ( A B L ) , u s i n g ferulic a c i d as a reference m a t e r i a l (26). C e l l - w a l l - e s t e r s of p - c o u m a r i c - ( P C ) a n d f e r u l i c - ( F A ) acids were h y d r o l y z e d w i t h 2 M N a O H a n d e s t i m a t e d after H P L C (26,32). M o n o m e r i c c o m p o s i t i o n s were o b t a i n e d f o l l o w i n g nitrobenzene o x i d a t i o n (32), or t h i o a c i d o l y s i s , w i t h gas c h r o m a t o g r a p h y - m a s s s p e c t r o m e t r y ( G C M S ) d e t e r m i n a t i o n of t h i o a c i d o l y s i s p r o d u c t s (33). L i g n o c e l l u l o s e ( L C ) , recovered after 5 % H 2 S O 4 p r e t r e a t m e n t (34), a n d s a p o n i f i c a t i o n residues ( S R ) , o b t a i n e d after N a O H h y d r o l y s i s of phenolic esters (26), were c h a r acterized a c c o r d i n g to the procedures p r e v i o u s l y adopted for c h a r a c t e r i z a t i o n of p a r i e t a l residue (32,33). P h e n o l i c acids ( p - c o u m a r i c , caffeic, ferulic a n d s i n a p i c ) were o b t a i n e d f r o m F L U K A a n d used w i t h o u t p u r i f i c a t i o n ; 5 - h y d r o x y f e r u l i c a c i d was a gift f r o m N . G . L e w i s ( V i r g i n i a P o l y t e c h n i c I n s t i t u t e a n d S t a t e U n i v e r s i t y , B l a c k s b u r g , V A 24061, U S A ) . Results and Discussion Lignification of Fescue. R e s u l t s s h o w n i n a l l T a b l e s are the m e a n of three d e t e r m i n a t i o n s . T a b l e s I a n d II show l i g n i n contents a n d m o n o m e r c o m p o s i t i o n s for b o t h n o r m a l a n d b r i t t l e fescue grass ecotypes. These were harvested at two l o c a t i o n s : G r i g n o n a n d L u s i g n a n . D a t a s h o w n were o n l y for one of two crops g r o w n at G r i g n o n , a n d one of three at L u s i g n a n . N o v i s i b l e differences between the same crops f r o m either l o c a t i o n were d i s cernible. In a l l cases, t h o u g h , l i g n i n contents were s i g n i f i c a n t l y higher at G r i g n o n t h a n at L u s i g n a n . A s p l a n t s were harvested at the same stage of development, differences can be ascribed to e n v i r o n m e n t a l effects. T a b l e I also shows the differences i n overall l i g n i n contents of b o t h ecotypes f r o m the same o r i g i n . Interestingly, o n l y a c e t y l b r o m i d e l i g n i n ( A B L ) contents were s i g n i f i c a n t l y different between fescues grown at G r i g n o n , w h i l e o n l y sulfuric a c i d l i g n i n s ( K L a n d A I L ) contents were different at L u s i g n a n ; such v a r i a t i o n s are difficult to e x p l a i n at present. F u r t h e r m o r e , i n each case, a c i d - i n s o l u b l e l i g n i n contents were lower t h a n K l a s o n l i g n i n contents c o n f i r m i n g the i m p o r t a n c e , i n the A I L procedure, of the 5 % s u l f u r i c a c i d p r e h y d r o l y s i s step required i n the case of green p l a n t s , w h i c h are u s u a l l y r i c h i n p r o t e i n as discussed p r e v i o u s l y (34). A B L contents, expressed i n ferulic a c i d equivalents, were of the same order of m a g n i t u d e as K l a s o n a n d a c i d - i n s o l u b l e l i g n i n contents. However, this agreement was f o r t u i t o u s as the A B L d e t e r m i n a t i o n p r o v i d e d o n l y

13.

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GAUDILLERE & MONTIES

T a b l e I. K l a s o n , A c i d - i n s o l u b l e , a n d A c e t y l b r o m i d e L i g n i n C o n t e n t s of N o r ­ m a l a n d B r i t t l e Fescue E c o t y p e s G r o w n at G r i g n o n a n d L u s i g n a n ( s t a n d a r d d e v i a t i o n less t h a n 10%) Lignin Content G r i g n o n Harvest

Where

(%) Lusignan Harvest

T y p e of Lignin

Normal (%)

Brittle (%)

Normal (%)

Brittle (%)

KL AIL ABL

20.4 14.0 18.0

19.4 14.4 15.5

17.2 11.2 12.0

15.1 7.7 13.0

KL = AIL = ABL =

Klason lignin A c i d Insoluble L i g n i n Acetyl Bromide Lignin

Table II. L i g n i n M o n o m e r C o m p o s i t i o n , O b t a i n e d by Nitrobenzene O x i d a ­ t i o n of L i g n i n f r o m N o r m a l a n d B r i t t l e Fescue G r o w n at L u s i g n a n ( S a m e as i n T a b l e I) Normal Mass %

PR

LC

PR

LC

V S V + s

1.2 0.4 1.6 0.3

0.8 0.5 1.3 0.6

1.3 0.4 1.7 0.3

0.8 0.4 1.2 0.5

s/ν Where

V S PR LC

= = = =

Brittle

Vanillin Syringaldehyde P a r i e t a l residue Lignocellulose

a m o u n t s r e l a t i v e to ferulic a c i d a b s o r p t i v i t y . Differences between A B L values of n o r m a l a n d b r i t t l e fescue must be related to v a r i a t i o n s i n l i g n i n c o n t e n t , since no significant differences i n the t o t a l content of p - c o u m a r i c a n d ferulic esters were f o u n d between samples f r o m the same o r i g i n ( d a t a not s h o w n ) . T h e m o n o m e r i c c o m p o s i t i o n of l i g n i n i n p a r i e t a l residues ( P R ) , a n d the c o r r e s p o n d i n g lignocellulose ( L C ) of n o r m a l a n d b r i t t l e fescue, h a r v e s t e d at L u s i g n a n are s h o w n i n T a b l e II. A s differences between K L a n d A I L c o n ­ tents h a d p r e v i o u s l y o n l y been f o u n d between fescues g r o w n at L u s i g n a n ( T a b l e I), the l i g n i n c o m p o s i t i o n of P R a n d the c o r r e s p o n d i n g L C f r a c t i o n s were c o m p a r e d . A s can be seen f r o m T a b l e II, no s i g n i f i c a n t differences between n o r m a l a n d b r i t t l e ecotypes were observed. H o w e v e r , s i g n i f i c a n t differences i n m o n o m e r i c c o m p o s i t i o n of l i g n i n f r o m L C a n d P R are c l e a r l y

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discernible for each ecotype. These differences m a y be due to a c i d pret r e a t m e n t , r e s u l t i n g i n differences i n condensation reactions a n d loss of a c i d - s o l u b l e l i g n i n fractions (34). A s s i m i l a r trends were observed for b o t h ecotypes, the results suggest a great s i m i l a r i t y between the r e a c t i v i t y of the l i g n i n s of b o t h p l a n t s . T h u s , comparisons of l i g n i n of b o t h ecotypes revealed t h a t differences were m a i n l y related to e n v i r o n m e n t a l factors, a n d not genetic v a r i a b i l i t y . Lignification of Maize Internodes. L i g n i n v a r i a b i l i t y i n maize was s t u d i e d b y c o m p a r i s o n of the l i g n i n contents a n d m o n o m e r i c c o m p o s i t i o n of its internodes. T a b l e III shows a weak t r e n d i n K L a n d A I L contents between the u p p e r a n d lower internodes of b o t h ecotypes. L i g n i n contents are s l i g h t l y higher i n the lower internode, i n agreement w i t h previous results (4). T h i s c o n c l u s i o n was further strengthened b y c o m p a r i s o n of the A B L ( S R ) d a t a . A s a l k a l i n e h y d r o l y s i s , used for the S R p r e p a r a t i o n , h a d p r e v i o u s l y o n l y s o l u b i l i z e d phenolic esters a n d a f r a c t i o n of the l i g n i n , t h i s d a t a confirmed not o n l y a higher l i g n i n content, b u t also a lower r e a c t i v i t y (delignification) of the l i g n i n core i n the lower internodes of b o t h types. A B L d a t a for p a r i e t a l residues ( P R ) are more difficult to interpret because they i n c l u d e b o t h l i g n i n a n d phenolic esters. These b o u n d esters differ for b o t h ecotypes. In agreement w i t h K u c et al. (20,21), o n l y ferulic a n d p - c o u m a r i c acids were f o u n d as the two m a i n phenolic esters l i n k e d to the cell walls of n o r m a l a n d m u t a n t m a i z e . In each case, the P C / F E r a t i o for n o r m a l m a i z e was a b o u t twice t h a t of the b . m . m u t a n t ( d a t a not s h o w n ) ; these results are i n agreement w i t h previous studies on b m - 1 , b u t not b m - 3 , m u t a n t s (20,21). Table III. Klason ( K L ) , Acid-insoluble ( A I L ) and Acetylbromide ( A B L ) L i g n i n C o n t e n t s i n U p p e r a n d L o w e r Internodes f r o m N o r m a l a n d b . m . M u t a n t of M a i z e . ( P R = p a r i e t a l residue, S R = N a O H s a p o n i f i c a t i o n residue, s t a n d a r d d e v i a t i o n less t h a n 10%) L i g n i n contents

(%)

Normal

K L (PR) AIL (PR) A B L (PR) A B L (SR)

Mutant

Upper

Lower

Upper

Lower

17.6 10.7 12.5 1.8

18.5 11.2 12.3 4.6

14 6.4 9.8 1.5

14.5 7.5 10.3 2.3

T a b l e s I V a n d V show the m o n o m e r c o m p o s i t i o n of l i g n i n s for b o t h p a r i e t a l a n d s a p o n i f i c a t i o n residues. In t h i s r e g a r d , c o m p a r i s o n between P R a n d S R values allows the c h a r a c t e r i z a t i o n of the l i g n i n core, w h i c h is not s o l u b i l i z e d after alkaline t r e a t m e n t (20,21). Instead of u s i n g n i t r o b e n zene o x i d a t i o n , t h i o a c i d o l y s i s was used to characterize the non-condensed

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Lignification of Gramineae

m o n o m e r i c u n i t s l i n k e d b y a r y l - a l k y l ether linkages i n the l i g n i n p o l y m e r s . T h i o a c i d o l y s i s allows a m o r e specific c h a r a c t e r i z a t i o n o f l i g n i n (35); i n the case of woods, i t provides a good c o r r e l a t i o n w i t h n i t r o b e n z e n e o x i d a t i o n d a t a (36) a n d , i n the case o f g r a m i n e a e , i t allows a n u n a m b i g u o u s d i s c r i m i n a t i o n between l i g n i n m o n o m e r i c u n i t s a n d associated p h e n o l i c acids. T h i s is not possible b y direct nitrobenzene o x i d a t i o n o f p a r i e t a l residues (37). A s s h o w n i n F i g u r e 1, the two m a i n t h i o a c i d o l y s i s p r o d u c t s of n o n condensed g u a i a c y l ( G ) a n d s y r i n g y l (S) u n i t s are clearly separable by gas c h r o m a t o g r a p h y w i t h relative r e t e n t i o n times (R ) of R f = 1.14, 1.15 a n d R f = 1.24, 1.25 w i t h reference to tetracosane as an i n t e r n a l s t a n d a r d . U n der these c o n d i t i o n s , the R t ' s of p - c o u m a r i c a c i d ( P C ) , ferulic a c i d ( F A ) a n d t h e i r a d d i t i o n p r o d u c t s w i t h e t h a n e t h i o l , were R = 0.69, R = 0.85, R = 0.77 a n d R = 0.81, respectively. T h u s , t h i o a c i d o l y s i s p r o d u c t s o f p h e n o l i c acids a n d l i g n i n m o n o m e r i c u n i t s can be clearly s e p a r a t e d ; t h i s is not the case for nitrobenzene o x i d a t i o n p r o d u c t s where, for e x a m p l e , v a n i l l i n c a n originate f r o m either l i g n i n m o n o m e r s or ferulic a c i d . t

P

FE

F

E

C

P

C

A

A

T a b l e I V . M o n o m e r i c C o m p o s i t i o n of L i g n i n i n the P a r i e t a l R e s i d u e ( P R ) of the U p p e r a n d L o w e r Internode f r o m S t e m or N o r m a l a n d b . m . - M u t a n t of M a i z e s h o w n b y T h i o a c i d o l y s i s ( G = g u a i a c y l a n d S = s y r i n g y l - t r i t h i o e t h y l e t h e r s : F i g . 1). ( Y i e l d s are expressed as m i c r o m o l e s per g r a m o f A B L i n each s a m p l e of P R ; s t a n d a r d d e v i a t i o n less t h a n 1 0 % ) Normal

Mutant

Upper

Lower

Upper

Lower

126 109 234 0.86

168 289 457 1.71

184 11 193 0.06

234 69 303 0.29

G S S + G S/G

T a b l e V . M o n o m e r i c C o m p o s i t i o n of L i g n i n i n the S a p o n i f i c a t i o n R e s i d u e ( S R ) of the U p p e r a n d L o w e r Internode f r o m S t e m of N o r m a l a n d b . m . M u t a n t of M a i z e as s h o w n b y T h i o a c i d o l y s i s ( a b b r e v i a t i o n s a n d d a t a as i n Table IV) Normal

Mutant

Upper

Lower

Upper

Lower

72 56 122 0.74

202 224 426 1.10

73 n.c. 73 n.c.

217 30 248 0.14

G S S + G S/V n . c : not c a l c u l a t e d .

188

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PN A j j i



PM

SN

lis

SM

Q F i g u r e 1. P a r t i a l G C c h r o m a t o g r a m s h o w i n g the peaks of the m a i n t h i o a c i d o l y s i s p r o d u c t s , separated as T M S derivatives i n f u n c t i o n of t i m e ( t ) , f r o m p a r i e t a l residue of n o r m a l ( P N ) a n d m u t a n t ( P M ) m a i z e a n d , f r o m c o r r e s p o n d i n g s a p o n i f i c a t i o n residue S N a n d S M . F o r each t y p e of m o n o m e r : g u a i a c y l ( G ) a n d s y r i n g y l (S), two erythro a n d threo T M S g l y c e r o l - t r i t h i o e t h y l e t h e r isomers were observed w i t h s i m i l a r mass s p e c t r a . T h e case o f X isomers, w i t h s i m i l a r f r a g m e n t a t i o n p a t t e r n s to those of G a n d S, has been discussed elsewhere (45).

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T a b l e I V shows t h a t , i n b o t h ecotypes, the y i e l d s of non-condensed g u a i a c y l a n d s y r i n g y l u n i t s were higher i n the lower i n t e r n o d e . Thus, l i g n i n w a s a p p a r e n t l y less condensed i n lower, r a t h e r t h a n u p p e r , i n t e r n odes. F r o m the s y r i n g y l to g u a i a c y l r a t i o i t was also e v i d e n t t h a t m o r e s y r i n g y l u n i t s were deposited i n the lower i n t e r n o d e . Interestingly, the m u t a n t s h a d s l i g h t l y higher g u a i a c y l contents a n d m u c h r e d u c e d s y r i n g y l contents, i n c o m p a r i s o n to the n o r m a l p l a n t . U s i n g n i t r o b e n z e n e o x i d a t i o n , K u c et al h a d r e p o r t e d e x a c t l y the opposite for n o r m a l a n d b . m . 3 - m u t a n t s of m a i z e (21). U s i n g their n i t r o b e n z e n e o x i d a t i o n m e t h o d for c o m p a r i s o n , the m o n o m e r i c c o m p o s i t i o n of the lower internodes f r o m m u t a n t a n d n o r m a l m a i z e were again d e t e r m i n e d . I n agreement w i t h t h i o a c i d o l y s i s d a t a , the m u t a n t showed the same trends as before. T h u s , the d i s c r e p a n c y between these results a n d d a t a f r o m K u c et ai (21) c a n n o t be a t t r i b u t e d to differences i n the a n a l y t i c a l procedures used, a n d a n a l t e r n a t e e x p l a n a t i o n is r e q u i r e d . In the case of s a p o n i f i c a t i o n residues ( T a b l e V ) , these trends were even m o r e p r o n o u n c e d for the u p p e r i n t e r n o d e . T o t a l y i e l d s o f t h i o a c i d o l y s i s p r o d u c t s , (S + G ) , were s l i g h t l y higher for the case o f P R ( T a b l e I V ) , t h a n t h a t o f S R ( T a b l e V ) . However, the r e s i d u a l l i g n i n f r o m the S R of the lower internodes was r e l a t i v e l y u n c h a n g e d w h e n c o m p a r e d w i t h P R , w i t h the difference between S R a n d P R b e i n g n e a r l y negligible i n the case of n o r m a l m a i z e b u t significant i n the case of the m u t a n t . C o m p a r i s o n of q u a l i t a t i v e a n d q u a n t i t a t i v e d a t a o n l i g n i f i c a t i o n of the u p p e r a n d the lower internodes of m a i z e stems s h o w n i n T a b l e s III to V , i n d i c a t e d o n l y weak q u a n t i t a t i v e differences i n l i g n i n contents. H o w e v e r , there were significant differences i n l i g n i n m o n o m e r i c c o m p o s i t i o n a n d rea c t i v i t y ( d e l i g n i f i c a t i o n ) between internodes i n the m a i z e s t a l k . T h e l i g n i n contents a n d m o n o m e r i c c o m p o s i t i o n s were also c o m p a r e d between fractions of internodes as follows: E a c h of the u p p e r a n d lower internodes p r e v i o u s l y s t u d i e d was d i v i d e d i n t o three p a r t s of e q u a l l e n g t h . C o m p a r i s o n o f l i g n i n contents a n d m o n o m e r i c c o m p o s i t i o n s o f u p p e r a n d lower p a r t s of each i n t e r n o d e revealed o n l y weak differences ( d a t a not shown). Conclusions R e s u l t s f r o m t h i s s t u d y suggest several differences i n l i g n i f i c a t i o n between ecotypes of fescue a n d m u t a n t s o f m a i z e . W h i l e weak differences i n t o t a l l i g n i n contents were observed between b r i t t l e fescue a n d n o r m a l p l a n t ecotypes, no significant v a r i a t i o n s i n l i g n i n m o n o m e r i c c o m p o s i t i o n a n d rea c t i v i t y were e v i d e n t , even after 5% H2SO4 p r e t r e a t m e n t . E n v i r o n m e n t a l effects o n l i g n i f i c a t i o n of fescues were m i n o r a n d m a i n l y changed o n l y l i g n i n contents; t h u s , i n contrast to rice, the b r i t t l e character of fescue was not due to direct v a r i a t i o n s i n l i g n i n content or m o n o m e r i c c o m p o s i t i o n . E v e n t h o u g h brittleness o f p l a n t organs was not measured a n d q u a n t i t a t i v e l y rel a t e d t o l i g n i f i c a t i o n p a r a m e t e r s , i t seems l i k e l y t h a t o r g a n b r i t t l e n e s s a n d l i g n i n content are not r e l a t e d . O n the other h a n d , a r e l a t i o n s h i p between brittleness a n d s t r u c t u r e of fibers a n d p o l y s a c c h a r i d e content has been re-

190

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p e a t e d l y suggested (24,38,39,40). L i k e l y the b r i t t l e properties rely u p o n the f o r m a t i o n of m o l e c u l a r associations a n d n e t w o r k s , whose properties d e p e n d m o r e o n m u t u a l a s s o c i a t i o n of components t h a n o n t h e i r i n t r i n s i c c h e m i c a l c o m p o s i t i o n . T h i s t o p i c was discussed recently (41) a n d w o u l d require réévaluation i n the case of b r i t t l e rice a n d fescue. U n l i k e the cases of rice a n d fescue, differences i n b o t h l i g n i n c o n tent a n d m o n o m e r i c c o m p o s i t i o n were f o u n d between n o r m a l p l a n t s a n d the b . m . 3 - t y p e m u t a n t o f m a i z e . I n agreement w i t h K u c et ai (20,21), l i g n i n contents were always lower i n these m u t a n t s . H o w e v e r , the s y r i n g y l / g u a i a c y l r a t i o s were s i g n i f i c a n t l y lower t h a n those described p r e v i o u s l y for the b . m . 3 - t y p e (21). A s i m i l a r discrepancy was also noted for the P C / F E r a t i o s . In b o t h cases, the ratios f o u n d for the b . m . 3 - t y p e were s i m i l a r to values r e p o r t e d for the b . m . l - t y p e by these a u t h o r s (20,21). T h e s e differences c a n n o t be e x p l a i n e d at present, b u t m a y be related t o the fact t h a t the b . m . genes, or genetic b l o c k s , were expressed i n a different epigenetic e n v i r o n m e n t i n the case o f K u c ' s e x p e r i m e n t s a n d i n the case r e p o r t e d here. Differences i n l i g n i f i c a t i o n between the m a i z e internodes m a y be rel a t e d t o the biosynthesis a n d elongation o f the s t e m (4); to our knowledge, n o d a t a have been p u b l i s h e d concerning the m u t a n t s o f m a i z e i n t h i s respect. In a d d i t i o n to e n v i r o n m e n t a l effects o n l i g n i n contents, significant differences were f o u n d between internodes i n terms of b o t h l i g n i n content a n d m o n o m e r i c c o m p o s i t i o n . W e a k differences were also f o u n d w i t h i n i n ternodes. S u c h differences m a y be e x p l a i n e d b y the b i o s y n t h e t i c m o d e l of g r o w t h of gramineae stems i n w h i c h i n t e r c a l a t i n g m e r i s t e m s s u b t e n d the development of a series of separated internodes. T h e b i o s y n t h e t i c heterogeneity of l i g n i n i n the a p i c a l internode o f wheat (reported p r e v i o u s l y (20,30)) are i n agreement w i t h t h i s m o d e l ; these differences are related not o n l y t o l i g n i n properties, b u t also to associated c e l l - w a l l - p h e n o l i c s i n t h e i r r e l a t i o n to cell w a l l r e t i c u l a t i o n (41,42). It s h o u l d be evident t h a t a n u n e q u i v o c a l e s t i m a t i o n o f the r e l a t i v e effects o f genetic a n d e n v i r o n m e n t a l factors o n gramineae l i g n i f i c a t i o n requires more b i o s y n t h e t i c studies i n r e l a t i o n to organs, tissues a n d cell diff e r e n t i a t i o n , a n d less g l o b a l b i o c h e m i c a l a n a l y s i s . In p a r t i c u l a r heterogeneity of l i g n i n (43) s h o u l d be e m p h a s i z e d . A n a t o m i c a l e x a m i n a t i o n of b . m . - m u t a n t of m a i z e , for e x a m p l e , has a l r e a d y i n d i c a t e d t h a t , possibly, several k i n d s o f l i g n i n s are f o u n d w i t h i n the same p l a n t i n different t i s sues (44). V e r y recently, d u r i n g the e d i t i n g of the m a n u s c r i p t , t h i o a c i d o l ysis of l i g n i n f r o m internodes of b . m . - m u t a n t has s h o w n t h a t a d d i t i o n a l 5h y d r o x y g u a i a c y l m o n o m e r i c u n i t s , c o m p o u n d X ( F i g . 1) were i n c o r p o r a t e d i n t o the l i g n i n of t h i s m u t a n t (45), c o n f i r m i n g the p o s s i b i l i t y of q u a l i t a tive v a r i a t i o n s i n l i g n i n w h i c h m a y be o f great interest for b i o t e c h n o l o g i c a l m a n i p u l a t i o n o f lignins. Acknowledgments T h e a u t h o r s are grateful to E l i s a b e t h G r e n e t a n d J . J a d a s - H e c a r t ( I N R A ) for p r o v i d i n g samples of respectively b . m . 3 m a i z e , b r i t t l e fescue a n d corr e s p o n d i n g n o r m a l p l a n t s ; t h a n k s are also due to D r . C a t h e r i n e L a p i e r r e

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for h e l p f u l discussions a n d c r i t i c a l review o f m a n u s c r i p t a n d t o F . J a g i c for p l a n t c u l t i v a t i o n at G r i g n o n . Literature Cited

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26. Sharma, U.; Brillouet, J. M.; Scalbert, Α.; Monties, B. Agronomie 1986, 6, 265-271. 27. Jadas-Hecart, J. Agronomie 1985, 5, 459-462. 28. Davies, I. Welsh Plant Breed. Stat. Tech. Bull. 1969, 3, 61-76. 29. Agossin, E.; Odier, E.; Gaudillere, M.; Monties, B. Bull. Groupe Polyphenols 1982, 11, 187-195. 30. Gaudillere, M.; Monties, B. Proc. Fourth Cell Wall Meet. (Cell Wall 86); Paris, 1986, 284-287. 31. Grenet, E.; Barry, P. Reprod. Nut. Dev. 1988, 28, 125-129. 32. Scalbert, Α.; Monties, B.; Rolando, C. B. Holzforschung 1986, 40, 119127. 33. Lapierre, C.; Monties, B.; Rolando, C. Holzforschung 1986, 40, 113118. 34. Monties, B. Agronomie 1981, 4, 317-321. 35. Rolando, C.; Lapierre, C.; Monties, B. In Methods in Lignin Chemistry; Lin, S. Y.; Dence, C. W., Eds.; Springer, in press. 36. Tollier, M-T.; Monties, B.; Lapierre, C.; Herve du Penhoat, C.; Rolando, C. Holzforschung 1986, 40(supp.), 75-79. 37. Lapierre, C.; Scalbert, Α.; Monties, B.; Rolando, C. Bull. Groupe Polyphenols 1986, 13, 128-135. 38. Kneebone, W. R. Agron. J. 1960, 52, 539-542. 39. Wilson, D. J. Agric. Sci. 1965, 65, 285-292. 40. Coley, P. D. Ecolog. Monog. 1983, 53, 209-233. 41. Monties, B. In Production et Utilisation des Biomasses Lignocellu­ losiques; Monties, B., Ed.; Apria-Lavoisier: Paris, in press. 42. Ranner, G. R.; Morrisson, J. M. J. Sci. Food Agric. 1983, 34, 137-144. 43. Monties, B. In The Biochemistry of Plant Phenolics (Ann. Proc. Phy­ tochem. Soc. Europ.); Van Sumere, C. F.; Lea, P. J., Eds.; Clarendon: Oxford, 1985; 25, 161-181. 44. Wardrop, A. B. In Proc. Int. Symp. Wood & Pulping Chem. (ISWPC); S.T.F.I. Pub.: Stockholm, 1981; I, 44-51. 45. Lapierre, C.; Tollier, M.-T.; Monties, B. C. R. Acad. Sci. Paris 1988, 307, 723-728. RECEIVED May 19, 1989