Plant Cell Wall Polymers - American Chemical Society

Chapter 3

Toward a Working Model of the Growing Plant Cell Wall Phenolic Cross-Linking Reactions in the Primary Cell Walls of Dicotyledons Stephen C . Fry and Janice G . Miller

Downloaded by UNIV LAVAL on July 12, 2016 | http://pubs.acs.org Publication Date: July 31, 1989 | doi: 10.1021/bk-1989-0399.ch003

Department of Botany, University of Edinburgh, The King's Buildings, Mayfield Road, Edinburgh E H 9 3 J H , Scotland

The efficient formation of inter-polymeric cross-links by oxidative coupling of the small number of polymer-bound phenolic side-chains present in the non-lignified, growing plant cell wall requires considerable specificity in the reactions concerned. In this paper we summarize some of the evidence that such specificity exists. We suggest that the cell wall is initially assembled by non-covalent interactions, especially involving the hydrogen-bonding of neutral xyloglucan chains to several microfibrils, thereby tethering these microfibrils. Oxidative coupling of other matrix polymers [acidic polysaccharides and/or basic glycoproteins] via their phenolic side-chains is seen as a subsequent wall-modification reaction whereby xyloglucan chains may be strapped to their current microfibrils so that the existing architecture is rendered more nearly permanent. Efficient strapping (i.e., fastening the maximum amount of material with fewest "buckles") requires chemical specificity—the formation of cross-links at appropriate sites. There is great specificity both in the biosynthetic reactions by which phenolic side-chains become attached to the wall polymers and also in the choice of phenolic partners and orientation during coupling. T h e g r o w i n g plant cell w a l l contains polymers w h i c h bear a s m a l l p r o p o r t i o n o f phenolic side-chains. These side-chains appear t o be subject in vivo to o x i d a t i v e phenolic c o u p l i n g a n d thus t o p a r t i c i p a t e i n c r o s s - l i n k i n g r e actions t h a t m a y be h i g h l y significant i n the c o n t r o l o f w a l l e x t e n s i b i l i t y (and therefore i n cell growth) a n d o f e n z y m i c d i g e s t i b i l i t y (1,2). For phenolic c r o s s - l i n k i n g t o b e b i o l o g i c a l l y effective, despite the presence o f o n l y low levels of phenolic c o m p o u n d s i n the p r i m a r y cell w a l l , the reactions 0097-6156/89/0399-0033$06.00/0 © 1989 American Chemical Society

Lewis and Paice; Plant Cell Wall Polymers ACS Symposium Series; American Chemical Society: Washington, DC, 1989.

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PLANT C E L L W A L L POLYMERS

must be a c c u r a t e l y a n d efficiently steered: they m u s t be specific. Here we s u m m a r i z e evidence t h a t the necessary specificity exists.


Hypothesis In order to t h i n k about the n a t u r e a n d consequences of cell w a l l p o l y mer phenolic c r o s s - l i n k i n g , we need a w o r k i n g m o d e l of the m o d e of as s e m b l y a n d the final s t r u c t u r e of the p r i m a r y cell w a l l . U n f o r t u n a t e l y , there is no u n i v e r s a l l y acceptable m o d e l : t h a t proposed b y A l b e r s h e i m a n d co-workers (3) is not now w i d e l y accepted because the p o s t u l a t e d i n t e r p o l y s a c c h a r i d e glycosidic bonds have not been d e m o n s t r a t e d (4); a n d the ' w a r p - w e f t ' m o d e l of L a m p o r t (5) rests o n the a s s u m p t i o n s t h a t extensin (i) forms a defined-porosity network (not proven); (ii) is o r i e n t a t e d a n t i c l i n a l l y to the cell surface [some evidence against (6)]; a n d (iii) is a m a j o r component of a l l p r i m a r y cell walls (not true). Major Structural Polymers of the Cell Wall. A w a l l m o d e l requires a de s c r i p t i o n of the m a j o r p o l y m e r s i n v o l v e d . T h e m a j o r p o l y m e r s of the grow i n g D i c o t y l e d o n cell w a l l are s h o w n , a p p r o x i m a t e l y to scale, i n F i g u r e 1. T h e y are described i n more d e t a i l elsewhere (2); i n brief, they are: 1. The microfibrillar cellulose. T h i s forms the skeletal scaffolding of the cell w a l l . M i c r o f i b r i l s are about 4 n m i n diameter (6) a n d are of i n d e terminate length. 2. The neutral hemicelluloses. These are x y l o g l u c a n i n D i c o t y l e d o n o u s p r i m a r y walls a n d p r i n c i p a l l y m i x e d - l i n k e d β-(1—»3), (1—*4)-D-glucans i n members of the G r a m i n a c e a e (grasses, i n c l u d i n g cereals). In i s o l a t i o n , x y l o g l u c a n is water-soluble, a l t h o u g h the molecule is a r e l a tively stiff rod a p p r o x i m a t e l y 150 to 1500 n m i n t o t a l contour l e n g t h (8). O n e x y l o g l u c a n h a d a measured a x i a l r a t i o of about 100 (9). In the i n t a c t cell w a l l , the x y l o g l u c a n is firmly a t t a c h e d to the surface of the cellulosic m i c r o f i b r i l s , p r i n c i p a l l y by h y d r o g e n - b o n d i n g (2,8,10-13), a l t h o u g h some more secure bonds m a y also be present (2,10). 3. The acidic polysaccharides. In D i c o t y l e d o n s , the m a j o r acidic p o l y s a c charides are the pectins ( p a r t i a l l y methyl-esterifled h o m o g a l a c t u r o nans a n d r h a m n o g a l a c t u r o n a n s ) a n d s m a l l e r a m o u n t s of a r a b i n o g l u c u r o n o x y l a n s (2). [In g r o w i n g grass cell walls, the a r a b i n o g l u c u r o n o x y lans u s u a l l y p r e d o m i n a t e over pectins.] 4. The basic extensins. These are h y d r o x y p r o l i n e - , l y s i n e - a n d t y r o s i n e r i c h glycoproteins consisting of r i g i d m o l e c u l a r rods about 80 n m l o n g (14,15), b e a r i n g short m o n o - to tetrasaccharide side-chains (2,14). W h e n n e w l y secreted they b i n d i o n i c a l l y to the a c i d i c polysaccharides of the cell w a l l a n d can be e x t r a c t e d w i t h cold salt solutions; later they become m u c h more resistant to s a l t - e x t r a c t i o n a n d are s a i d to be covalently b o u n d , p r o b a b l y v i a d i m e r i z a t i o n of their tyrosine residues to f o r m isodityrosine (15). Some of the acidic a n d basic polymers of the cell w a l l bear pheno lic side-chains. T h e acidic polysaccharides carry ferulic a n d p - c o u m a r i c acid a n d related c i n n a m a t e - d e r i v a t i v e s , esterified to specific h y d r o x y groups


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Cellulose m i c r o f i b r i l

Xyloglucan molecule

Extensin molecule

scale 100 nm

F i g u r e 1. T h e p r i n c i p a l s t r u c t u r a l c o m p o n e n t s o f the g r o w i n g cell walls o f a D i c o t y l e d o n . T h e d r a w i n g s are a p p r o x i m a t e l y t o scale.


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


(1,2,17-22). F e r u l o y l a t e d pectins have been found i n the p a r e n c h y m a tous cell walls of m a n y D i c o t y l e d o n s ( m a i n l y i n the C e n t r o s p e r m a e a n d Solanaceae), b u t UV-fluorescence microscopy suggests t h a t at least the epidermal cell walls of a l l D i c o t y l e d o n s c o n t a i n phenolic residues; i t r e m a i n s to be seen whether these phenolic residues are a t t a c h e d to polysaccharides or to c u t i n , b u t l o c a t i o n of even a s m a l l q u a n t i t y of, say, f e r u l o y l - p e c t i n i n the e p i d e r m a l w a l l w o u l d be p a r t i c u l a r l y significant i n the c o n t r o l of g r o w t h because the e x t e n s i b i l i t y of the epidermis controls the e x p a n s i o n of whole stems (23) a n d leaves ( F r y , u n p u b l i s h e d observations). T h e extensins, as a l r e a d y m e n t i o n e d , are r i c h i n the phenolic a m i n o a c i d t y r o s i n e (2). Assembly of a Xyloglucan-Microfibril Framework to the Wall. T h e i n i t i a l assembly of the cell w a l l is l i k e l y t o be v i a non-covalent b o n d i n g , especially h y d r o g e n - b o n d i n g . These i n i t i a l steps are believed to be n o n - e n z y m i c . T h e m i c r o f i b r i l s are synthesized b y enzyme complexes w h i c h are m o b i l e i n the p l a s m a m e m b r a n e . T h e complexes c h u r n out n a k e d m i c r o f i b r i l s , w h i c h come to lie i n a p l a n e — t h e i n n e r m o s t , accreting face of the cell w a l l . A t the same t i m e , G o l g i - d e r i v e d vesicles deposit the essentially soluble m a t r i x p o l y m e r s ( F i g . 2a). E v i d e n c e suggesting t h a t the m a t r i x p o l y m e r s are indeed water-soluble when n e w l y secreted was p r o v i d e d by observation of the polysaccharides secreted b y n a k e d protoplasts d i r e c t l y into the c u l t u r e m e d i u m (24). O f the various m a t r i x p o l y m e r s i n t h i s m i x t u r e ( h e m i c e l luloses, pectins a n d e x t e n s i n ) , it is p a r t i c u l a r l y the x y l o g l u c a n t h a t w i l l s t r o n g l y h y d r o g e n - b o n d to the m i c r o f i b r i l s , c l o t h i n g t h e m w i t h a m o l e c u l a r monolayer (10-12). Since the t o t a l contour length of a x y l o g l u c a n molecule is 40 t o 400 times greater t h a n the diameter of a m i c r o f i b r i l (8), it w o u l d seem i n e v i t a b l e t h a t most of the newly deposited x y l o g l u c a n chains w i l l have the o p p o r t u n i t y to b i n d to the several m i c r o f i b r i l s across w h i c h they are r a n d o m l y l a i d d o w n ( F i g . 2b) (25). A n y tendency for a x y l o g l u c a n molecule to re-orientate a n d come to lie w i t h its entire length a l o n g a single m i c r o f i b r i l w i l l be m i n i m i z e d by the presence i n the space between the adjacent m i c r o f i b r i l s of other m a t r i x p o l y m e r s w h i c h are not s t r o n g l y h y d r o g e n - b o n d i n g (pectins a n d extensins) a n d w h i c h w i l l (a) r e t a r d the m o l e c u l a r m o t i o n of the x y l o g l u c a n a n d (b) [by some of t h e m l y i n g w i t h their m a i n - c h a i n s p e r p e n d i c u l a r to the m i crofibrils] p h y s i c a l l y prevent the x y l o g l u c a n chain f r o m l i n i n g up a l o n g a n y favored m i c r o f i b r i l . T h e consequence of t h i s is t h a t the i n d i v i d u a l x y l o g l u can molecules w i l l come t o be hydrogen-bonded a l o n g discrete segments of several m i c r o f i b r i l s , t e t h e r i n g t h e m , and the i n t e r v e n i n g lengths of x y l o g l u can w i l l be suspended between the m i c r o f i b r i l s ( F i g . 2b). It m a y be noted p a r e n t h e t i c a l l y t h a t evidence t h a t extensins are not s t r o n g l y hydrogen-bonded i n the cell w a l l is the ease w i t h w h i c h they can be leached out of the w a l l w i t h 25-50 m M L a C l 3 or AICI3 ( 2 U ) — a t r e a t m e n t t h a t effectively breaks ionic bonds but not hydrogen b o n d s — s o l o n g as the t r e a t m e n t is a p p l i e d before the extensin has become covalently b o n d e d i n the cell w a l l . E v i d e n c e t h a t at least one pectic p o l y s a c c h a ride ( r h a m n o g a l a c t u r o n a n - I ) is not s t r o n g l y h y d r o g e n - b o n d e d i n the cell


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3.

F i g u r e 2. A s p e c u l a t i v e m o d e l for the assembly a n d g r o w t h o f the D i c o t y l e donous cell w a l l . F o r c l a r i t y , the m o d e l shows t w o m i c r o f i b r i l s = = = , = = = ) a n d o n l y one of the m a n y x y l o g l u c a n molecules ( ) t h a t are p r o p o s e d to interconnect t h e m . T h e loops i n the lower d i a g r a m represent other m a t r i x p o l y m e r s ( e x t e n s i n a n d / o r pectins) whose p h e n o l i c side-chains have b e c o m e o x i d a t i v e l y c o u p l e d . Steps (d) a n d (g) are p r o p o s e d t o be c a t a l y z e d b y cellulase. F o r f u r t h e r d e t a i l s , see ref. (25).


38



w a l l is the fact t h a t , despite its large size [ D P 2,000 (27); s i m i l a r to a n average x y l o g l u c a n ] , i t is released i n t a c t f r o m the cell w a l l i n t o c o l d n e u t r a l water following t r e a t m e n t w i t h pure e n d o - p o l y g a l a c t u r o n a s e , a n e n z y m e t h a t hydrolyzes h o m o g a l a c t u r o n a n ( w i t h w h i c h the backbone o f the r h a m n o g a l a c t u r o n a n h a d p r e s u m a b l y been contiguous) b u t does not affect the r h a m n o g a l a c t u r o n a n itself. An Important Requirement for Cell Growth. W i t h continued cell e x p a n s i o n , the i n t e r m i c r o f i b r i l l a r segments of x y l o g l u c a n w i l l become stretched t a u t ( F i g . 2c) a n d w i l l e v e n t u a l l y come to bear the b u r d e n of t u r g o r . F u r t h e r g r o w t h w i l l then be impossible unless one or b o t h of two t h i n g s h a p p e n s : 1. the i n t e r - m i c r o f i b r i l l a r segments of x y l o g l u c a n are h y d r o l y z e d ( F i g . 2d) b y a n endo-/?-(l—>4)-D-glucanase (cellulase), a well-established p l a n t cell w a l l enzyme (28); 2. the xyloglucan-cellulose hydrogen-bonds are p h y s i c a l l y p u l l e d o p e n , u n z i p p i n g the x y l o g l u c a n molecules f r o m the m i c r o f i b r i l s ( F i g . 2e); hydrogen-bonds are considerably weaker t h a n the covalent b o n d s of w h i c h the i n d i v i d u a l polysaccharide chains are c o n s t r u c t e d (13). Consequences and Requirements of Oxidative Phenolic Coupling in the Cell Wall. If the phenolic side-chains present o n pectins a n d / o r extensins c a n cross-link i n a n a p p r o p r i a t e m a n n e r a n d place, they c o u l d f o r m loops t h a t w o u l d encircle m i c r o f i b r i l s a n d thereby strap p a r t i c u l a r x y l o g l u c a n molecules on to p a r t i c u l a r m i c r o f i b r i l s ( F i g . 2f). T h i s w o u l d prevent the " u n z i p p i n g " m o d e of g r o w t h , b u t w o u l d not affect the " h y d r o l y t i c " m o d e . T h u s , w h i l e c r o s s - l i n k i n g c o u l d p o t e n t i a l l y decelerate g r o w t h very r a p i d l y ( F i g . 2 h ) , such a n effect need not be irreversible ( F i g . 2g). T h e i m p o r t a n c e of t h i s conclusion can be considered by reference to a p h y s i o l o g i c a l e x a m ple: T h e r a p i d l y - i m p o s e d i n h i b i t o r y effect of blue light o n the g r o w t h of stems has been hypothesized to be m e d i a t e d b y peroxidase-catalyzed crossl i n k i n g of w a l l p o l y m e r - b o u n d phenolics (29); since the i n i t i a l g r o w t h rate is r a p i d l y restored when the blue light is s w i t c h e d off, the hypothesis o n l y stands i f the m e c h a n i s m of g r o w t h i n h i b i t i o n is reversible. Efficient cross-link f o r m a t i o n by a s m a l l n u m b e r of w a l l p o l y m e r - b o u n d phenolics requires great precision i n the m e t a b o l i c reactions i n v o l v e d . It is not sufficient to f o r m cross-links: the cross-links need to be formed i n the p r o p e r place w i t h i n the p o l y m e r molecule a n d w i t h i n the cell w a l l . E v i d e n c e t h a t cross-links f o r m at a l l [albeit sometimes as a low percentage of the t o t a l w a l l phenolics] is presented elsewhere (1,2,13,16,30-32). Here we present evidence t h a t sufficient m o l e c u l a r specificity exists to be c o m p a t i b l e w i t h useful cross-link f o r m a t i o n . E v i d e n c e for Specificity i n t h e R e a c t i o n s G r o u p s are I n t r o d u c e d i n t o W a l l P o l y m e r s

by

which

Phenolic

Background. In order to m a x i m i z e the efficiency of c r o s s - l i n k i n g based o n a s m a l l number of phenolic groups, i t is i m p o r t a n t t h a t these groups s h o u l d be sited o n the w a l l p o l y m e r s at appropriate loci rather t h a n r a n d o m l y . In the case of the phenolic side-chains of extensins this c r i t e r i o n is met since the s i t i n g of the tyrosines is genetically encoded (27).



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Siting of Ferulic Acid in the Wall Polysaccharides. T h e o r i g i n of the feru l o y l residues of the a c i d i c w a l l polysaccharides is not so s t r a i g h t f o r w a r d . H o w e v e r , there is g o o d evidence t h a t these phenolic side-chains are very specifically s i t e d . T h e p o s i t i o n of the f e r u l o y l groups can most easily be e x p l o r e d b y e n z y m i c " d i s s e c t i o n " of the polysaccharides w i t h c o m m e r c i a l m i x t u r e s of enzymes such as t h a t k n o w n as Driselase (available f r o m S i g m a C h e m i c a l C o . ) . Driselase, w h i c h is w i d e l y used for the i s o l a t i o n o f p l a n t p r o t o p l a s t s because i t possesses enzymes t h a t h y d r o l y z e most of the g l y cosidic linkages of the p r i m a r y cell w a l l , lacks feruloyl-esterase a c t i v i t y and therefore leaves the f e r u l o y l groups a t t a c h e d to the a p p r o p r i a t e sugar u n i t of the p o l y s a c c h a r i d e . I n a d d i t i o n , the endo- a n d exo-glycanases of Driselase cannot h y d r o l y z e those glycose residues t h a t bear f e r u l o y l g r o u p s . T h e expected p r o d u c t s of h y d r o l y s i s of a f e r u l o y l - p o l y s a c c h a r i d e are t h u s m a i n l y monosaccharides p l u s feruloyl-disaccharides (2). [Driselase l a c k s α-xylosidase a c t i v i t y a n d therefore also y i e l d s large a m o u n t s of a s i m p l e d i s a c c h a r i d e , D - x y l o p y r a n o s y l - a - ( l —• 6)-D-glucose, f r o m xyloglucan.] T h e cell walls of D i c o t y l e d o n s , especially i n the C a r y o p h y H a l e s , y i e l d u p o n Driselase digestion two m a j o r f e r u l o y l - d i s a c c h a r i d e s , n a m e l y 3-0-(3-0-feruloyl-a-L-arabinopyranosyl)-L-arabinose (Fer-Ara2) and 4 - 0 (6-0-feruloyl-/?-D-galactopyranosyl)-D-galactose ( F e r - G a l ) (17). These two c o m p o u n d s together account for 6 0 - 7 0 % of the f e r u l o y l residues of the p r i m a r y walls of c u l t u r e d s p i n a c h cells. In the grasses, the m a j o r f e r u l o y l - d i s a c c h a r i d e o b t a i n e d w i t h Driselase is 3 - 0 - ( 5 - 0 - f e r u l o y l a - L - a r a b i n o f u r a n o s y l ) - D - x y l o s e ( F e r - A r a - X y l ) , b u t larger a m o u n t s of a feruloyl-trisaccharide, 4-0-[3-0-(5-0-feruloyl-a-L-arabinofuranosyl) -/?-Dx y l o p y r a n o s y l ] - D - x y l o s e ( F e r - A r a - X y l ) , u s u a l l y p r e d o m i n a t e because D r i selase is inefficient at h y d r o l y z i n g x y l o b i o s e (19,20). 2

2

T h e i m p o r t a n t conclusion is t h a t m u c h of the w a l l ' s ferulic a c i d is l i n k e d to specific h y d r o x y groups o n specific sugars of specific p o l y s a c c h a r i d e s . T h e specificity is p a r t i c u l a r l y n o t a b l e i n the case of F e r - A r a , since the f e r u l o y l a t e d arabinose residues are i n the rare pyranose r i n g - f o r m (17). It is clear t h a t the f e r u l o y l a t i o n reactions are not r a n d o m , b u t are carefully steered b i o s y n t h e t i c steps. 2

Evidence for Intracellular Feruloylation Reactions. T h e reactions by w h i c h f e r u l o y l residues are added to polysaccharides o c c u r i n t r a c e l l u l a r l y . T h i s was established for the F e r - A r a u n i t s of the pectic polysaccharides of c u l ture s p i n a c h cells b y a d m i n i s t r a t i o n of [ H]arabinose so t h a t the careers of p o l y m e r - b o u n d pentose residues c o u l d be followed f r o m t h e i r i n t r a c e l l u l a r i n c o r p o r a t i o n i n t o nascent polysaccharides a n d g l y c o p r o t e i n s , v i a t h e i r se c r e t i o n t h r o u g h the p l a s m a m e m b r a n e , to t h e i r u l t i m a t e fate w i t h i n the w a l l (28). T h e cells were fed [ H]arabinose for various defined periods of t i m e , after w h i c h two d i s t i n c t analyses were p e r f o r m e d ( F i g . 3): a. A s a m p l e of the cells was k i l l e d i n e t h a n o l a n d the a l c o h o l - i n s o l u b l e residue ( c o n t a i n i n g the polysaccharides a n d g l y c o p r o t e i n s ) was d i gested e x h a u s t i v e l y w i t h Driselase. T h i s converted the various pentosec o n t a i n i n g u n i t s of the p o l y m e r s to the f o l l o w i n g m a j o r b r e a k d o w n products: 2

3

3


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[3H]Arabinose

POLYMERS

Ethanol

Driselase

Washings rejected

Digestion products


time t

Spinach cells

Culture filtrate

- - Polymers

Origin—!

O -

-Xyl-a-(l-6)Glc

o -

-Ara

o -

-xyi

o —

--Fer-Ara2

Ι Λ Λ Λ Λ Λ / V V V V V V N F i g u r e 3. Scheme o f a n e x p e r i m e n t to d e m o n s t r a t e the i n t r a c e l l u l a r n a t u r e of polysaccharide-feruloylation.


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arabinan feruloyl-arabinan arabinoxylan xyloglucan

—• [ —• [ —• [ —• [

3

3

3

3

41

H]arabinose H]arabinose + Fer-[ H]Ara2 H]arabinose + [ H]xylose + [ H]xylobiose H ] x y l o s y l - a - ( l —• 6) glucose 3

3

3

— w h i c h were t h e n separated b y paper c h r o m a t o g r a p h y a n d assayed i n d i v i d u a l l y for H b y s c i n t i l l a t i o n - c o u n t i n g , b . A s a m p l e of the c u l t u r e filtrate was also a n a l y z e d b y p a p e r c h r o m a t o g r a p h y to separate the u n c h a n g e d [ H ] a r a b i n o s e f r o m a n y secreted p o l y s a c c h a r i d e present. T h e H i n the l a t t e r ( R p = 0.00) was m e a s u r e d by scintillation-counting. B y step (a), a p i c t u r e c o u l d be formed o f the k i n e t i c s o f the i n t r a c e l l u lar i n c o r p o r a t i o n of H f r o m [ H ] a r a b i n o s e i n t o the m a j o r polysaccharides o f the cell w a l l . R a d i o a c t i v i t y was i n c o r p o r a t e d i n t o p o l y m e r - b o u n d a r a binose a n d xylosyl-glucose u n i t s after l a g periods of a b o u t 3.5 m i n a n d 6.5 m i n , respectively. These lags are taken t o be the t i m e r e q u i r e d for the [ H ] a r a b i n o s e t o be taken u p a n d activated t o the a p p r o p r i a t e d o n o r for p o l y s a c c h a r i d e biosynthesis 3

3

3


3

3

3

[ H]Ara — [ H]Ara-l-P ~ 3

3

UDP-[ H]Ara (~ 3

UDP-[ H]Xyl) 3

(1,35) a n d p o s s i b l y for the U D P - s u g a r s to be t r a n s p o r t e d i n t o the e n d o m e m b r a n e s y s t e m where the polysaccharides are s y n t h e s i z e d . A f t e r the l a g , the rate of i n c o r p o r a t i o n of H i n t o p o l y m e r s r e m a i n e d f a i r l y constant for several h o u r s . T h e i n c o r p o r a t i o n of H f r o m [ H ] a r a b i n o s e i n t o F e r - A r a 2 u n i t s showed a l a g o f a b o u t 4.2 m i n , after w h i c h i t too b e c a m e l i n e a r . T h i s means t h a t as l i t t l e as 0.7 m i n after their i n c o r p o r a t i o n i n t o a ( p o s s i b l y s t i l l nascent) p o l y s a c c h a r i d e , [ H ] a r a b i n o s e residues were susceptible t o f e r u l o y l a t i o n . T h e f e r u l o y l a t i o n r e a c t i o n is t h u s l i k e l y to have been o c c u r r i n g i n the e n d o m e m b r a n e s y s t e m , c o - s y n t h e t i c a l l y , as one o f the h i g h l y r e g u l a t e d p a r t s o f the s o p h i s t i c a t e d p o l y s a c c h a r i d e - b i o s y n t h e t i c m a c h i n e r y . T h i s c a n be c o m p a r e d to the c o - t r a n s l a t i o n a l m o d i f i c a t i o n k n o w n t o o c c u r i n m a n y proteins. B y step (b), further evidence was o b t a i n e d t h a t the f e r u l o y l a t i o n was intracellular. T h e e x t r a c e l l u l a r polysaccharides a n d g l y c o p r o t e i n s o n l y s t a r t e d to acquire L after a l a g of a b o u t 25 m i n (34). T h i s is i n t e r preted t o m e a n t h a t before 25 m i n essentially a l l the [ H ] p o l y s a c c h a r i d e was s t i l l i n t r a c e l l u l a r , either i n the G o l g i bodies or packaged i n t o G o l g i d e r i v e d vesicles, b u t not yet passed t h r o u g h the p l a s m a m e m b r a n e . T h e fact t h a t these sugar residues were b e i n g f e r u l o y l a t e d , at the m a x i m a l r a t e , w e l l before 25 m i n s u p p o r t s the conclusion t h a t f e r u l o y l a t i o n was l a r g e l y intracellular. 3

3

3

3

3

3

Possible Extracellular Feruloylation. It has been suggested (36) t h a t feru l o y l a t i o n o c c u r s e x t r a c e l l u l a r l y . E v i d e n c e i n s u p p o r t of t h i s c o n t e n t i o n was the observation t h a t i n m a t u r i n g coleoptiles of b a r l e y the a m o u n t of p o l y s a c c h a r i d e - b o u n d ferulate continued t o increase for at least one d a y after t o t a l p o l y s a c c h a r i d e a c c u m u l a t i o n h a d ceased. However, t h i s fact is o p e n t o the a l t e r n a t i v e e x p l a n a t i o n t h a t r e l a t i v e l y s m a l l a m o u n t s o f a h i g h l y


42


f e r u l o y l a t e d polysaccharide continued to be synthesized ( i n t r a c e l l u l a r l y ) i n m a t u r e coleoptiles even after t o t a l w a l l polysaccharide synthesis h a d decelerated a n d been a p p r o x i m a t e l y equalled b y p o l y s a c c h a r i d e b r e a k d o w n so t h a t the net rate of polysaccharide a c c u m u l a t i o n was zero. F u r t h e r evidence a p p a r e n t l y i n favor of some f e r u l o y l a t i o n b e i n g ext r a c e l l u l a r was the observation t h a t [ C ] f e r u l o y l - C o A w i l l b i n d covalently to coleoptile cell walls i n vitro (37). It was suggested t h a t t h i s was due to the o p e r a t i o n of a n e x t r a c e l l u l a r feruloyltransferase c a t a l y z i n g a t r a n s esterification r e a c t i o n whereby the feruloyl residue is transferred f r o m the C o A donor to a polysaccharide acceptor. However, i t r e m a i n s t o be seen (i) whether f e r u l o y l - C o A , the p r o p o s e d donor, occurs e x t r a c e l l u l a r l y ; a n d (ii) whether, i n the in-vitro s y s t e m , the C r e m a i n s as [ C ] f e r u l o y l residues or whether the [ C ] f e r u l o y l residues become b o u n d t o other w a l l - b o u n d (non-radioactive) phenolic groups, perhaps b y o x i d a t i v e c o u p l i n g . [ O x i d a tive c o u p l i n g i n isolated cell walls i n the absence of a d d e d H 2 O 2 has been observed (38).] S u c h a reaction w o u l d be i n h i b i t e d b y ascorbate, c y a n i d e or p o s s i b l y catalase (31). If [ C ] f e r u l o y l - C o A does indeed b i n d to cell w a l l s in vitro v i a the f o r m a t i o n of ester b o n d s , it w i l l be of great interest t o see whether the C can be recovered b y Driselase digestion i n the f o r m of [ C ] F e r - A r a - X y l a n d [ C ] F e r - A r a - X y l i n d i c a t i n g t h a t the h i g h l y specific f e r u l o y l - s u g a r b o n d characteristic of grass cell walls h a d been synthesized b y a n e x t r a c e l l u l a r enzyme s y s t e m . B i o s y n t h e s i s of ester b o n d s i n the cell w a l l has a precedent i n the proposed biosynthesis of c u t i n f r o m f a t t y a c y l C o A thioesters i n the e p i d e r m a l cell w a l l (for a review, see 39). It is possible t h a t Y a m a m o t o et ai (37) have detected the biosynthetic s y s t e m b y w h i c h feruloyl residues are a t t a c h e d to the a l i p h a t i c core of c u t i n rather t h a n to the w a l l polysaccharides. T h e c u r r e n t evidence seems to favor i n t r a c e l l u l a r f e r u l o y l a t i o n of polysaccharides. 14

1 4

14


14

14

1 4

1 4

1 4

2

E v i d e n c e for Specificity i n t h e O x i d a t i v e C o u p l i n g o f P h e n o l i c S i d e - C h a i n s i n the C e l l W a l l T h e previous section has presented evidence t h a t phenolic u n i t s are caref u l l y positioned w i t h i n the w a l l p o l y m e r s . W h e n these u n i t s undergo oxi d a t i v e phenolic c o u p l i n g reactions i n the cell w a l l , the c o u p l i n g reactions themselves are also r e m a r k a b l y specific. T h i s can be i l l u s t r a t e d b y reference to the tyrosine residues of e x t e n s i n . Orientation of Coupling in Protein-Bound Tyrosine Residues. T y r o s i n e can couple to f o r m either of two isomeric d i m e r s , dityrosine a n d i s o d i t y r o s i n e (1,16) ( F i g . 4). T h e choice between these two dimers is governed b y the c o n d i t i o n s under w h i c h the c o u p l i n g is carried o u t ; some examples are given i n T a b l e I. In a n i m a l s t r u c t u r a l proteins in vivo, the o n l y k n o w n d i m e r of tyrosine is d i t y r o s i n e (40,41); i n the extensin of plant cell walls, i n contrast, the o n l y d i m e r formed in vivo is isodityrosine (16). H o w is the c o u p l i n g of t y r o s i n e i n plants confined to the f o r m a t i o n of isodityrosine? T h e r e is n o t h i n g u n i q u e about the l o c a l e n v i r o n m e n t of the tyrosine residues i n (pure) e x t e n s i n , since


3.

FRY & MILLER

Working Model of Growing Plant Cell Watt

43


T a b l e I. D i m e r i c P r o d u c t s of the O x i d a t i v e C o u p l i n g of P r o t e i n - B o u n d T y rosine Residues under V a r i o u s C o n d i t i o n s Substrate

System

PH

Products

Reference

R e s i l i n in vivo C o l l a g e n in vivo E x t e n s i n in vivo E x t e n s i n in vitro Bovine serum a l b u m i n in vitro E x t e n s i n in vitro

insect cuticle nematode cyst D i c o t p r i m a r y cell w a l l peroxidase + H 2 O 2

> 7 >7 < 7 9

DÎT DiT Idt DiT

(40) (41) (16) (38)

9 6

DiT Idt

(42) (38)

peroxidase + H 2 O 2 isolated cell w a l l

OH

OH

1OH

I

kJ ÇH CHNH 2

2

COOH Tyrosine

Κ

ÇH ÇHNH COOH 2

2

•tyrosine

Isodityrosine

F i g u r e 4. S t r u c t u r e s of t y r o s i n e , d i t y r o s i n e a n d i s o d i t y r o s i n e . [ R e p r o d u c e d w i t h p e r m i s s i o n f r o m Journal of Expenmental Botany 38, 853-62; © O x f o r d U n i v e r s i t y P r e s s , 1987.

these residues are q u i t e capable of f o r m i n g d i t y r o s i n e (38) under the same o p t i m a l in vitro c o n d i t i o n s as w o r k for v i r t u a l l y a n y other p r o t e i n (e.g., b o v i n e s e r u m a l b u m i n ) (42). T h e o n l y in vitro s y s t e m i n w h i c h i s o d i t y r o s i n e p r o d u c t i o n p r e d o m i n a t e s is i n e x t e n s i n treated w i t h i s o l a t e d p l a n t cell w a l l s (38) at p H 6. T w o factors associated w i t h the p l a n t cell w a l l were considered w h i c h m i g h t direct exclusive i s o d i t y r o s i n e f o r m a t i o n : a. The pH of the plant cell wall T h i s p H is always l i k e l y t o be b e l o w 7.0 in vivo, a n d therefore w e l l below the o p t i m u m ( p H ca. 9.0) for t o t a l d i m e r i z a t i o n of tyrosine; p H 9.0 was used i n most in vitro assays other t h a n those where cell walls were the source of peroxidase. b . Specific isoperoxidases. T h e cell w a l l c o n t a i n s a n u m b e r o f peroxidase isozymes, q u i t e d i s t i n c t f r o m the m a j o r basic i s o z y m e o b t a i n e d f r o m horseradish a n d used i n most in vitro assays; i t is possible t h a t some of these other isozymes have a p r o p e n s i t y to c a t a l y z e i s o d i t y r o s i n e formation.


44

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

Evidence Against Cell Wall pH and Isoperoxidase Specificity as Determi nants of Isodityrosine Formation. T o test these possible e x p l a n a t i o n s for the exclusive f o r m a t i o n of isodityrosine i n the p l a n t cell w a l l , samples of [ C ] t y r o s i n e were o x i d i z e d b y H 2 O 2 i n the presence of three s h a r p l y c o n t r a s t i n g horseradish-isoperoxidases [two l o w - p i (acidic) a n d one h i g h - p i ( b a sic)] at a wide range of p H values (37). T h e two l o w - p i isozymes were considerably poorer t h a n the h i g h - p l isozyme at c a t a l y z i n g t o t a l t y r o s i n e - o x i d a t i o n [the c o m p a r i s o n was based o n the use of a constant 1.2 / / k a t / m l of each isozyme, 1 / i k a t b e i n g the a m o u n t t h a t w i l l catalyze the o x i d a t i o n of p y r o g a l l o l to p u r p u r o g a l l i n at 1 μ η ι ο ΐ / s at p H 6.0 a n d 2 5 ° C ] . T h e l o w e r - p i isozyme o x i d i z e d tyrosine o p t i m a l l y at p H 6, a n d the h i g h - p i isozyme h a d an o p t i m u m of p H 9. T h e rate of o x i d a t i o n by the h i g h - p i isozyme at p H 9 was a b o u t seven t i m e s greater t h a n t h a t of the l o w e r - p l isozyme at p H 6. T h i s confirms t h a t the isozymes e x h i b i t e d considerable differences i n c a t a l y t i c properties, as well as differing i n p i .


14

W h e n the d i m e r s p r o d u c e d b y these isozymes, at a w i d e range of ρ H values, were a n a l y z e d i n d i v i d u a l l y , no great difference was f o u n d (37). A t h i g h p H values ( p H 8-10), b o t h the l o w - p l a n d h i g h - p i isozymes generated ca. 20 times more dityrosine t h a n i s o d i t y r o s i n e . A s the p H was lowered, the y i e l d of i s o d i t y r o s i n e increased a n d t h a t of d i t y r o s i n e decreased u n t i l at p H 3 there was o n l y about 2-3 times more d i t y r o s i n e t h a n i s o d i t y r o s i n e ; however, under no c o n d i t i o n s d i d the y i e l d of i s o d i t y r o s i n e ever exceed t h a t of d i t y r o s i n e . It m a y be concluded t h a t neither p H nor isozyme-specificity is l i k e l y t o direct the exclusive f o r m a t i o n of i s o d i t y r o s i n e i n the p l a n t cell w a l l i n vivo. Indeed, i t m i g h t be argued t h a t isozyme-specificity is i n t r i n s i c a l l y u n l i k e l y t o direct the o r i e n t a t i o n of c o u p l i n g ( d i t y r o s i n e vs. isodityrosine) since the role of the enzyme is thought to be merely the p r o d u c t i o n of t y r o s i n e free r a d i c a l s (44) w h i c h t h e n non-enzymically p a i r off. Role of Neighboring Polysaccharide Molecules in Determining the Orienta tion of Tyrosine Residues During Coupling. These considerations suggest a t h i r d possible e x p l a n a t i o n for the exclusive f o r m a t i o n o f i s o d i t y r o s i n e i n the p l a n t cell w a l l i n vivo: t h a t the n e i g h b o r i n g s t r u c t u r a l molecules of the w a l l c o n s t r a i n extensin to prevent dityrosine f o r m a t i o n . T h i s w o u l d m e a n t h a t the b i o l o g i c a l l y relevant s u b s t r a t e for peroxidase i n the p l a n t cell w a l l is not n a k e d e x t e n s i n b u t extensin complexed w i t h another w a l l c o m p o nent, p o s s i b l y a n acidic polysaccharide to w h i c h the e x t e n s i n w o u l d b i n d ionically. Conclusions In c o n c l u s i o n , i t seems fair to say t h a t specificity exists i n b o t h the b i o s y n thesis a n d i n the o x i d a t i v e c o u p l i n g of p o l y m e r - b o u n d phenols i n the grow i n g cell w a l l , (a) T y r o s i n e residues are placed at specific sites a l o n g the extensin molecule b y genetically-encoded i n f o r m a t i o n , (b) T y r o s i n e crossl i n k i n g i n vivo is a very specific, carefully steered process i n t h a t i t occurs


3.

FRY & M I L L E R

Working Model ofGrowing Plant CellWall

45

o n l y w h e n t h e extensin is i n a precise molecular e n v i r o n m e n t , p o s s i b l y as a n i o n i c c o m p l e x w i t h a n a c i d i c p o l y s a c c h a r i d e . T h i s i s evidenced b y t h e fact t h a t cell walls couple their t y r o s i n e residues t o m a k e i s o d i t y r o s i n e r a t h e r t h a n d i t y r o s i n e , whereas t h e same residues i n t h e absence o f a cell w a l l generate m a i n l y d i t y r o s i n e . (c) T h e f e r u l o y l a t i o n a n d p - c o u m a r o y l a t i o n of a c i d i c polysaccharides occurs o n h i g h l y specific h y d r o x y g r o u p s , (d) I t r e m a i n s t o be seen h o w precise o r r a n d o m t h e c o u p l i n g o f p o l y s a c c h a r i d e b o u n d p h e n o l i c side-chains i s .


T h e significance o f t h i s precision is t h a t i t suggests t h a t adequate s p e c i ficity exists for t h e c o u p l i n g reactions t o take p a r t efficiently i n t h e " s t r a p p i n g " o f hemicellulose molecules t o m i c r o f i b r i l s m e n t i o n e d earlier. It w i l l b e of great interest i n future research t o explore w h e t h e r a n d t o w h a t extent such " s t r a p p i n g " occurs. A cknowledgment s W e are grateful t o t h e A g r i c u l t u r a l a n d F o o d Research C o u n c i l for t h e a w a r d o f a research g r a n t i n s u p p o r t o f o u r w o r k . Literature Cited

1. Fry, S. C. Ann. Rev. Plant Physiol. 1986, 37, 165-86. 2. Fry, S. C. The Growing Plant Cell Wall: Chemical and Metabolic Anal ysis; Longman: London; and Wiley: New York, 1988. 3. Keegstra, K.; Talmadge, K. W.; Bauer, W. D.; Albersheim, P. Plant Physiol. 1973, 51, 188-96. 4. Darvill, A. G.; McNeil, M.; Albersheim, P.; Delmer, D. P. In The Bio chemistry of Plants: A Comprehensive Treatise; Vol. 1; Preiss, J., Ed.; Academic Press: New York, 1980; pp. 91-162. 5. Lamport, D. T. A. In Cellulose: Structure, Modification and Hydrol ysis; Young, R. Α.; Rowell, R. M., Eds.; Wiley: New York, 1986; pp. 77-90. 6. Stafstrom, J. P.; Staehelin, L. A. Planta 1988, 174, 321-32. 7. Rubin, G. C. This volume. 8. Fry, S. C. J. Exp. Bot. 1989, 40, 1-11. 9. O'Neill, R. Α.; Selvendran, R. R. Carbohydr. Res. 1983, 111, 239-55. 10. Hayashi, T.; Marsden, M. P. F.; Delmer, D. P. Plant Physiol. 1987, 83, 384-9. 11. MacKay, A. L.; Wallace, J. C.; Sasaki, K.; Taylor, I. E. P. Biochemistry 1988, 27, 1467-73. 12. MacKay, A. L.; Wallace, J. C.; Sasaki, K.; Taylor, I. E. P. This volume. 13. Fry, S. C. Mod. Meth. Plant Analysis, New Series, 10, in press. 14. Heckman, J. W.; Terhune, B. T.; Lamport, D. T. A. Plant Physiol. 1988, 86, 848-56. 15. Stafstrom, J. P.; Staehelin, L. A. Plant Physiol. 1986, 81, 234-41. 16. Fry, S. C. Biochem. J. 1982, 204, 449-55. 17. Fry, S. C. Biochem. J. 1982, 203, 493-504. 18. Fry, S. C. Planta 1983, 157, 111-23.


46

19. 20. 21. 22. 23. 24. 25. 26.


27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43. 44.

PLANT C E L L W A L L

POLYMERS

Kato, Y.; Nevins, D. J. Carbohydr. Res. 1985, 137, 139-50. Ahluwalia, B.; Fry, S. C. J. Cer. Sci. 1986, 4, 287-95. Smith, M. M.; Hartley, R. D. Carbohydr. Res. 1983, 118, 65-80. Gubler, F.; Ashford, A. E.; Bacic, Α.; Blakeney, A. B.; Stone, B. A. Ausi. J. Plant Physiol. 1985, 12, 307-17. Sachs, J. Handbuch der Experimentalphysiologie der Pflanzen; Engelmann: Leipzig, 1865. Hanke, D. E.; Northcote, D. H. J. Cell Sci. 1974, 14, 29-50. Fry, S. C. Physiol. Plant. 1989, 75, in press. Smith, J. J.; Muldoon, E. P.; Lamport, D. T. A. Phytochemistry 1984, 23, 1233-40. McNeil, M.; Darvill, A. G.; Fry, S. C.; Albersheim, P. Ann. Rev. Biochem. 1984, 53, 625-63. Hayashi, T.; Wong, Y.; Maclachlan, G. Plant Physiol. 1984, 75, 605-10. Shinkle, J. R.; Jones, R. L. Plant Physiol. 1988, 86, 960-6. Fry, S. C. Phytochemistry 1984, 23, 59-64. Fry, S. C.; Miller, J. G. Food Hydrocolloids 1987, 1, 395-7. Biggs, K. J.; Fry, S. C. In Physiology of Cell Expansion During Plant Growth; Cosgrove, D. J.; Knievel, D. P., Eds.; Am. Soc. Plant Physiol.; 1987; pp. 46-57. Chen, J.; Varner, J. E. EMBO J. 1985, 4, 2145-51. Fry, S. C. Planta 1987, 171, 205-11. Fry, S. C.; Northcote, D. H. Plant Physiol. 1983, 73, 1055-61. Yamamoto, E.; Towers, G. H. N. J. Plant Physiol. 1985, 117, 441-9. Yamamoto, E.; Bokelman, G. H.; Lewis, N. G., this volume. Cooper, J. B.; Varner, J. E. Plant Physiol. 1984, 76, 414-7. Holloway, P. J. In The Plant Cuticle; Cutler, D. F.; Alvin, K. L.; Price, C. E., Eds.; Academic: London, 1982; pp. 45-85. Andersen, S. O. Biochim. Biophys. Acta 1964, 93, 213-5. Lopez-Llorca, L. V.; Fry, S. C. Nematologica 1989, in press. Aeschbach, R.; Amado, R.; Neukom, H. Biochim. Biophys. Acta 1976, 439, 292-301. Fry, S. C. J. Exp. Bot. 1987, 38, 853-62. Ralston, I. M.; Dunford, H. B. Can. J. Chem. 1980, 58, 1270-6.

RECEIVED May 19, 1989


Plant Cell Wall Polymers - American Chemical Society

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