Plant Cell Wall Polymers - American Chemical Society

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

6

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

13.

GAUDILLERE & MONTIES

Lignification of Gramineae

183

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

184

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.

185



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

186


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

13.


187


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


PN A j j i

1Λ

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).

13.



189

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


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

13.



191

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

1. Jouany, J. P.; Besle, J. M. In Production et Utilisation des Biomasses Lignocellulosiques; Monties, B., Ed.; Apria-Lavoisier: Paris, 1988 (in press). 2. Anonyme. In Informations-Βois; AFOCEL: Paris, 1988, p. 7, 15. 3. Akin, D. E. In Nutritional Limits to Animal Production from Pastures; Hacker, J. B. Ed.; CSIRO Pub., Comm. Agric. Bur., Farnham Royal (UK), 1981; p. 201-223. 4. Hacker, J. B.; Minson, D. J. Herbage Abstr. 1981, 51, 459-482. 5. S-Thiago, L. R. L.; Kellaway, R. C. Anim. Feed. Sci. Technol. 1982, 7, 71-81. 6. Aman, P.; Nordkvist, E. Swedish J. Agric. Res. 1983, 13, 61-67. 7. Sullivan, J. T. J. Anim. Sci. 1959, 18, 1292-1298. 8. Lindberg, J. E.; Ternrud, I. E.; Theander, O. J. Sci. Food Agric. 1984, 35, 500-506. 9. Jung, H. J.; Fahey, G. C. Jr. J. Anim. Sci. 1983, 57, 206-219. 10. Reeves, J. B., Jr. J. Anim. Sci. 1985, 60, 316-322. 11. Wilson, J. R. In Nutritional Limits to Animal Production from Pas tures; Hacker, J. B., Ed.; CSIRO pub., Comm. Agr. Bur., Farnham Royal (UK), 1981; p. 112-131. 12. Norton, B. W. In Nutritional Limits to Animal Production from Pas tures; Hacker, J. B., Ed.; CSIRO pub., Comm. Agr. Bur., Farnham Royal (UK), 1981; p. 89-110. 13. Hacker, J. B. In Nutritional Limits to Animal Production from Pas tures; Hacker, J. B., Ed.; CSIRO pub., Comm. Agr. Bur., Farnham Royal (UK), 1981; p. 305-326. 14. Morrisson, I. M. Grass Forage Sci. 1980, 35, 287-293. 15. Hartley, R. D. J. Sci. Food Agric. 1971, 23, 1347-1354. 16. Crosthwaite, C.; Ishimara, M.; Richards, G. N. J. Sci. Food Agric. 1984, 35, 1041-1050. 17. Jackson, M. G. Anim. Feed Sci. Technol. 1977, 2, 105-130. 18. Morrisson, I. M. J. Sci. Food Agric. 1988, 42, 295-304. 19. White, L. M.; Hartman, G. P.; Bergman, J. W. Agron. J. 1981, 73, 117-121. 20. Kuc, J.; Nelson, Ο. E. Arch. Biochem. Biophys. 1964, 105, 103-113. 21. Kuc, J.; Nelson, Ο. E.; Flanagan, P. Phytochem. 1968, 7, 1435-1436. 22. Porter, K. S.; Axtell, J. D.; Lechtenberg, V. L.; Colenbrander, V. F. Crop Science 1978, 18, 205-208. 23. Bulcholtz, D. L.; Cantrell, R. P.; Axtell, J. D.; Lechtenberg, V. L. J. Agric. Food Chem. 1980, 28, 1239-1241. 24. Doat, J.; Marie, R. Ann. Amelior. Plantes 1977, 27, 705-715. 25. Monties, B.; Mestres, C.; Bagdhadi, K. In Proc. First Intl. Symp. Wood & Pulp. Chem. (ISWPC); S.T.F.I. Pub.: Stockholm, 1981; 5, 40-43.

192

PLANT C E L L W A L L

POLYMERS

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

Plant Cell Wall Polymers - American Chemical Society

Recommend Documents