Chapter 14
Inhibition of Cell Wall Peroxidases with Ferulic Salts and Fluorinated Analogues 1
1
2
Anne-Marie Catesson , An Pang , Charlette Francesch , Christian Rolando , and Renée Goldberg 2
1
1
Biomembranes et Surfaces Cellulaires Végétales, ENS, 46 rue d'Ulm, 75230 Paris Cedex 05, France
2
Laboratoire de l'Activation Moléculaire, ENS, 24 rue Lhomond, 75231 Paris Cedex 05, France
The extent of inhibition of the oxidation of peroxidase substrates by ferulic salts was quite variable, from no inhibition to total inhibition. Total inhibition occurred when the substrate (e.g., syringaldazine) was closely related to ferulic acid. The presence of afluorineatom in ferulic acid slightly reduced the inhibitory effect. Oxidation of ferulic compounds was restricted to lignifying cell walls in situ. Cell wall peroxidases from bark and xylem were fractionated into their component isozymes. Two main anionic groups were present in the xylem and their activity towards ferulic salts and their fluorinated analogues was determined. Whether the two isozymes represent enzymes specifically involved in lignin biosynthesis is discussed. Peroxidases are w i d e l y d i s t r i b u t e d enzymes i n the p l a n t k i n g d o m . D e s p i t e their u b i q u i t y a n d the ever-increasing n u m b e r o f f u n c t i o n s a s c r i b e d t o t h e m , the precise role a n d l o c a l i z a t i o n o f the m a n y isozymes r e m a i n u n c e r t a i n (1,2). O n e o f the difficulties o f peroxidase studies is t h a t t h e enzymes c a n react w i t h a n u m b e r o f s y n t h e t i c or n a t u r a l substrates a n d t h a t even t h e use of p u r i f i e d isozymes i n assays for substrate specificity does n o t i d e n t i f y a n y definitive roles. A search for specific i n h i b i t o r s represents another a p p r o a c h w h i c h is s t i l l p o o r l y developed despite i t s p o t e n t i a l u t i l i t y . T h e o n l y b i o l o g i c a l f u n c t i o n w h i c h has been r e p e a t e d l y confirmed i s the role o f peroxidases i n l i g n i n m o n o m e r p o l y m e r i z a t i o n (1). B u t even i n t h i s case, the role o f the various isozymes is n o t yet clear, a l t h o u g h a n i o n i c , cell w a l l b o u n d peroxidases generally seem t o be i n v o l v e d i n l i g n i f i c a t i o n (1,2). I n p l a n t cell walls, l i g n i n m o n o m e r s seem t o be present in vivo i n the f o r m o f c i n n a m y l alcohols. In vitro, their a c i d precursors c a n also b e o x i d i z e d b y peroxidases (3). I n order t o g a i n further insight i n t o the possible 0097-6156/89/0399~0193$06.00A) © 1989 American Chemical Society
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role of each isoperoxidase i n l i g n i n biosynthesis, we decided to check the p o t e n t i a l i n h i b i t o r y effects of c i n n a m y l c o m p o u n d s a n d their fluorinated analogues. T h e exchange of h y d r o g e n for a fluorine a t o m i n the 2 p o s i t i o n of the propane c h a i n (^-carbon) s h o u l d interact w i t h the site of c o u p l i n g of l i g n i n m o n o m e r s . I n t h i s r e g a r d , a p r e l i m i n a r y survey (4) showed t h a t fluorinated c i n n a m y l alcohols a n d acids h a d s i m i l a r effects t o u n f l u o r i n a t e d c o m p o u n d s . However, a l t h o u g h alcohols are n a t u r a l l i g n i n precursors, they are difficult to use as i n h i b i t o r s since they are water i n s o l u b l e . H e n c e , a d e t a i l e d s t u d y was u n d e r t a k e n o n the a c t i o n of the acids. T h e present p a p e r reports c o m p a r a t i v e results o b t a i n e d w i t h ferulic a n d fluoroferulic acids o n cell w a l l peroxidases. Materials and
Methods
F i v e - to s i x - m o n t h - o l d tobacco plants (Nicotiana tabacum v a r . S a m s u n ) g r o w n i n a glasshouse at 20° C were used for t h i s s t u d y . C o m m e r c i a l s y n t h e t i c substrates e m p l o y e d b o t h for h i s t o c h e m i c a l a n d b i o c h e m i c a l as says were g u a i a c o l , p - p h e n y l e n e d i a m i n e - p y r o c a t e c h o l ( P P D - P C ) , 3-3' d i aminobenzidine ( D A B ) , tetramethylbenzidine ( T M B ) and syringaldazine. I s o p r o p y l a m i n e a n d m o n o s o d i u m salts of ferulic a c i d were also used as s u b strates as w e l l as t h e i r " ^ - f l u o r i n a t e d analogues" s u b s t i t u t e d w i t h a fluorine a t o m o n the /?-carbon ( F i g . 1). H i s t o c h e m i c a l observations were done o n h a n d - m a d e transverse sections of fresh t o b a c c o stems. B i o c h e m i c a l assays were p e r f o r m e d separately o n b a r k (inner c o r t i c a l p a r e n c h y m a , p h l o e m a n d fibres) a n d x y l e m fractions. T e c h n i c a l d a t a of i n c u b a t i o n , e n z y m e e x t r a c t i o n , s p e c t r o p h o t o m e t r y a n d electrophoretic assays were given elsewhere (5-7). Synthesis of fluorinated c o m p o u n d s was p e r f o r m e d as p r e v i o u s l y described (4). Results and Discussion Oxidation of Salts from Ferulic and β-Fluoroferulic Acids. W h e n stem sections were i n c u b a t e d w i t h ferulic a c i d , i s o p r o p y l a m i n e or s o d i u m s a l t s , the cell walls of the youngest x y l e m or s c l e r e n c h y m a elements were s t a i n e d a light p i n k color. N o reaction was observed i n other cell walls (Table I). T h e same result was o b t a i n e d w i t h fluorinated analogues. T h e fact t h a t o n l y peroxidases f r o m l i g n i f y i n g cell walls are able to oxidize ferulic c o m p o u n d s a n d s y r i n g a l d a z i n e must be e m p h a s i z e d . A b s o r p t i o n s p e c t r a of the p i n k o x i d a t i o n p r o d u c t s of ferulic a c i d a n d /?-fluoroferulic a c i d i n the presence of hydrogen peroxide a n d peroxidases e x t r a c t e d f r o m tobacco cell walls ("covalently b o u n d " fraction) showed a peak at 520 n m . T h u s ferulic a c i d , w h i c h is not in vivo a n a t u r a l substrate for p e r o x i dases i n v o l v e d i n l i g n i f i c a t i o n processes, can be o x i d i z e d not o n l y in vitro b u t also in situ, i.e., i n the n o r m a l , biological e n v i r o n m e n t of the e n z y m e . F u r t h e r m o r e , the o x i d a t i o n seems to be l i m i t e d to the walls of l i g n i f y i n g cells. T h i s r e s t r i c t e d l o c a l i z a t i o n has been described o n l y i n the case of sy r i n g a l d a z i n e , a s y n t h e t i c s u b s t r a t e closely related to c i n n a m i c c o m p o u n d s (8,9). It is i n t e r e s t i n g to note t h a t the presence of a fluorine a t o m o n the
14.
CATESSON E T AL.
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Inhibition of Cell Wall Peroxidases
T a b l e I. S t a i n i n g R e l a t i v e Intensity O b s e r v e d i n T o b a c c o S t e m Sections I n c u b a t e d w i t h Different P e r o x i d a s e S u b s t r a t e s . Transverse sections were i n c u b a t e d w i t h H 2 O 2 a n d a peroxidase s u b s t r a t e . T h e o x i d a t i o n o f r o u t i n e l y used c o m m e r c i a l substrates was checked i n the presence or the absence of ferulic a c i d salts. V a r i a t i o n s of s t a i n i n g i n t e n s i t y i n cell walls (— t o + + ) were j u d g e d by o b s e r v a t i o n w i t h a light microscope
Substrate Ferulic Salt /?-Fluoroferulic Salt Syringaldazine 4-Ferulic S a l t +/?-Fluoroferulic Salt
Parenchyma
—
-
TMB +Ferulic Salt +/?-Fluoroferulic Salt
+
DAB +Ferulic Salt +/?-Fluoroferulic Salt
++ + + ++ +
Guaiacol -hFerulic Salt +/?-Fluoroferulic Salt PPD-PC +Ferulic Salt +/?-Fluoroferulic Salt
-
-/+ -/+ -/+
Bark Fibres
+ + ++ -
-/+ ++ -/+ -/+ ++ + -/+ ++ + + ++ +/++ +/++
Phloem
Young X y l e m
-
+ + ++
++ -
++ +/++ -/+ ++ -/+ -/+ -/+ -/+ -/+
-
-/+ ++ -/+ -/+ ++ + + ++ -/+ + ++ +/++ +/++
/?-carbon does not alter the r e s u l t s . T h i s suggests t h a t the red color m a y be due t o reactions oc c u r i n g o n the a r o m a t i c r i n g since the fluorine a t o m is present o n the side c h a i n . T h e s e d a t a agree w i t h the i d e a t h a t l i g n i f y i n g w a l l s c o n t a i n specific isozymes, each possessing different affinities towards given s u b s t r a t e s . I n order t o check t h i s h y p o t h e s i s , a n a n a l y s i s of cell w a l l peroxidase f r a c t i o n s was u n d e r t a k e n . Oxidative Activities of Cell Wall Isozymes. P r e v i o u s e l e c t r o p h o r e t i c s t u d ies (4) have revealed the presence of several isozymes i n a l l e n z y m e ext r a c t s . W e a t t e m p t e d therefore to f r a c t i o n a t e t h e m b y a n i o n exchange c h r o m a t o g r a p h y . W e began o u r s t u d y w i t h the most a c t i v e enzymes, i.e., the l i g h t l y - b o u n d ( " i o n i c a l l y - b o u n d " ) peroxidases f r o m b a r k tissues a n d the s t r o n g l y b o u n d ( " c o v a l e n t l y - b o u n d " ) enzymes f r o m the x y l e m . C h r o m a t o g r a m s o b t a i n e d w i t h b a r k i o n i c a l l y b o u n d peroxidases are s h o w n i n F i g u r e 2. E n z y m e a c t i v i t i e s were e s t i m a t e d w i t h i s o p r o p y l a m i n e s a l t f r o m /?-fluoroferulic a c i d , s y r i n g a l d a z i n e a n d T M B , respectively. T w o m a i n p e a k s
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COOH
F i g u r e 1. C h e m i c a l s t r u c t u r e o f ferulic a c i d (left) a n d /?-fluoroferulic a c i d (right).
F i g u r e 2. A n i o n exchange c h r o m a t o g r a m of i o n i c a l l y b o u n d p h l o e m p e r oxidases o n D E A E - S e p h a r o s e . C o l l e c t e d fractions were a n a l y z e d for t h e i r oxidase a c t i v i t y t o w a r d s T M B (1), s y r i n g a l d a z i n e (2) a n d i s o p r o p y l a m i n e s a l t f r o m /?-fluoroferulic a c i d (3). B o : c a t i o n i c peroxidases; B i a n d B 2 : a n i o n i c peroxidases. C o l u m n was e q u i l i b r a t e d w i t h 0.01 M p h o s p h a t e buffer ( p H 7.1). F r a c t i o n s were e l u t e d w i t h a N a C l g r a d i e n t (0-0.5 M ) i n the same buffer (0.01 M p h o s p h a t e , p H 7.1).
14.
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Inhibition of Cell Wall Peroxidases
( B i a n d B 2 ) o f a n i o n i c isozymes were separated a n d t h e i r p u r i t y checked b y p o l y a c r y l a m i d e gel electrophoresis. T h e y p r o b a b l y represented the t w o a n i o n i c groups r e p o r t e d i n crude e x t r a c t s of t o b a c c o tissues (2). B o t h p e a k s o x i d i z e d the three tested substrates b u t reactions were m a r k e d l y weaker w i t h the fluorinated s a l t . L a r g e a m o u n t s o f c a t i o n i c peroxidases ( B o ) were present i n the e x t r a c t . T h e y were also able to o x i d i z e the three s u b s t r a t e s . A c c o r d i n g to M a d e r (2), basic isozymes w o u l d represent c y t o p l a s m i c e n z y m e s b i n d i n g to the w a l l d u r i n g cell breakage. Ion exchange c h r o m a t o g r a p h y o f x y l e m e x t r a c t s allowed the s e p a r a t i o n of two a n i o n i c p e a k s ( X i a n d X 2 ) o f peroxidase a c t i v i t y w h e n T M B was used as s u b s t r a t e ( F i g . 3). O n c e the four a n i o n i c fractions were i s o l a t e d ( B i , B 2 , Χ ι , X 2 ) ) t h e i r a c t i v i t i e s were i n v e s t i g a t e d u s i n g ferulic or /?-fluoroferulic i s o p r o p y l a m i n e salts as s u b s t r a t e s . R a t e s were p l o t t e d as a f u n c t i o n of s u b s t r a t e c o n c e n t r a t i o n . T h e L i n e w e a v e r - B u r k p l o t s o b t a i n e d ( F i g . 4) were not a l w a y s s t r i c t l y l i n e a r as a l r e a d y r e p o r t e d i n the case of f e r u l i c a c i d a n d s c o l o p e t i n o x i d a t i o n (10,11). A n e s t i m a t i o n was m a d e of the a p p a r e n t K u s i n g the l i n e a r p a r t of the p l o t s a n d results were c o m p a r e d w i t h those o b t a i n e d for T M B . T h e values f o u n d i n t h i s case were i n the same order of m a g n i t u d e , a b o u t 0.5 x 10~" to 1 χ 1 0 ~ M . I n a l l e x t r a c t s , /?-fluoroferulic salt i n h i b i t e d e n z y m e a c t i v i t y for concentrations higher t h a n 0.25 χ 1 0 " M . m
3
3
2
Inhibition of Commercial Synthetic Substrates with Salts from Ferulic and β-Fluoroferulic Acids. T a b l e I s u m m a r i z e s the results o b t a i n e d o n s t e m sections i n c u b a t e d i n a m e d i u m c o n t a i n i n g one of the u s u a l c o m m e r c i a l s u b s t r a t e s a n d a salt of ferulic or /?-fluoroferulic a c i d . T h r e e types of i n t e r a c t i o n s c o u l d be observed: 1. N o i n h i b i t i o n , or o n l y a very slight one, c o u l d be seen o n sections i n c u b a t e d i n a P P D - P C m e d i u m . A b s o r p t i o n s p e c t r a d i d not show m u c h difference w h e n ferulic or /?-fluoroferulic a c i d were a d d e d to the assay m i x t u r e . 2. A r a t h e r weak i n h i b i t i o n was observed w h e n sections were i n c u b a t e d in D A B and guaiacol. 3. A s t r o n g i n h i b i t i o n o c c u r e d w i t h T M B a n d s y r i n g a l d a z i n e . F o r i n stance, s y r i n g a l d a z i n e o x i d a t i o n c o u l d be c o m p l e t e l y i n h i b i t e d w i t h ferulic a c i d either in situ ( T a b l e I) or in vitro. I n h i b i t i o n of s y r i n g a l d a z i n e a n d T M B o x i d a t i o n s was n o t i c e a b l y weaker w h e n /?-fluoroferulic salt was used i n s t e a d of ferulic s a l t . C o n s e q u e n t l y , at a g i v e n c o n c e n t r a t i o n a n i n h i b i t i o n c a n develop be tween ferulic a c i d (or their fluorinated analogues) a n d some of the c u r r e n t l y used s y n t h e t i c substrates b u t there is no i n t e r a c t i o n w i t h others, e.g., P P D P C . T h e same differences i n i n h i b i t o r y a b i l i t y were p r e v i o u s l y observed w i t h /?-fluoroconiferyl a l c o h o l (4). These differences m i g h t be r e l a t e d t o the c o n f i g u r a t i o n of substrate molecules. T h e strongest i n h i b i t i o n was observed w h e n the t h r e e - d i m e n s i o n a l s t r u c t u r e of the s u b s t r a t e was closely r e l a t e d t o c i n n a m y l c o m p o u n d s . T h i s suggests t h a t l i g n i f y i n g cell walls c o n t a i n at least one d i s t i n c t i v e isoperoxidase specifically able to recognize t h i s t y p e
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F i g u r e 3. A n i o n exchange c h r o m a t o g r a m of covalently b o u n d x y l e m p e r o x idases. C o l l e c t e d fractions were a n a l y s e d for their a c t i v i t y towards T M B . X i a n d X 2 : a n i o n i c peroxidases. F o r e l u t i o n d e t a i l s , see F i g u r e 2.
14.
CATESSON ET A L
Inhibition of Cell Wall Peroxidases
199
o f molecule. E x p e r i m e n t s w i t h /?-fluoroferulic salts c o r r o b o r a t e t h i s i n t e r p r e t a t i o n since the
fluorinated
analogue is less r e a d i l y o x i d i z e d w i t h the
specific isoperoxidase(s). T h e a c t i v i t y of isoperoxidases able to o x i d i z e f e r u l i c a c i d is not res t r i c t e d to a few s i m i l a r substrates such as s y r i n g a l d a z i n e or T M B . A w i d e r range of substrates are i n v o l v e d (3). S y r i n g a l d a z i n e - o x i d a s e s f r o m p o p l a r a n d s y c a m o r e are k n o w n to react at least w i t h T M B , P P D - P C a n d g u i a c o l , b u t t h e i r affinity t o w a r d s the last substrate is 100 to 1000 t i m e s lower t h a n for s y r i n g a l d a z i n e (5,7,9). S i m i l a r results were o b t a i n e d w i t h o t h e r m a t e r i als (12-14). H o w e v e r , other isozymes are not able to react w i t h ferulic a c i d or s y r i n g a l d a z i n e (5,7,12,13). Several hypotheses c o u l d be given to e x p l a i n o u r results. T h e first one postulates the s i m u l t a n e o u s existence of at least two active sites o n isozymes. O n e of t h e m w o u l d e x c l u s i v e l y recognize c i n n a m i c c o m p o u n d s a n d w o u l d be r e s t r i c t e d to isozymes i n v o l v e d i n l i g n i f i c a t i o n processes. A t the present t i m e , we are not aware of any e x p e r i m e n t a l a r g u m e n t s s u p p o r t i n g t h i s i n t e r p r e t a t i o n . A c c o r d i n g to a second h y p o t h e s i s , a l l isozymes w o u l d be able to o x i d i z e a w i d e range of substrates b u t t h e i r s p e c i f i c i t y t o w a r d s a given s u b s t r a t e w o u l d be m o d u l a t e d b y c e l l u l a r events p r o b a b l y t h r o u g h c o n f o r m a t i o n a l changes. T h i s i n t e r p r e t a t i o n agrees w i t h the h y pothesis of a p o l y f u n c t i o n a l i t y of peroxidase p r o t e i n m o d u l a t e d b y m e t a l ions (14). P h e n o l s were also p r o p o s e d as inducers of c o n f o r m a t i o n a l changes (15). Differences i n redox p o t e n t i a l between peroxidase isozymes m i g h t also e x p l a i n t h e i r specificity. I n t e r a c t i o n s between isozymes m i g h t also p l a y a role (16). Conclusions It is often s t a t e d t h a t the last step of l i g n i n biosynthesis requires the s i m u l t a n e o u s presence i n the cell w a l l of a specific isoperoxidase a n d i t s t w o s u b s t r a t e s , i.e., h y d r o g e n peroxide a n d l i g n i n m o n o m e r s . X y l e m a n d scler e n c h y m a cells are p r o g r a m m e d to synthesize these substances i n the course of t h e i r d i f f e r e n t i a t i o n . In fact, the presence of H 2 O 2 i n the cell w a l l was d e m o n s t r a t e d o n l y i n two instances, d u r i n g cell w a l l l i g n i f i c a t i o n (5,8) a n d d u r i n g p r i m a r y cell w a l l r e t i c u l a t i o n t h r o u g h p h e n o l i c b o n d s (17). T h u s H 2 O 2 p r o d u c t i o n a n d l i g n i n m o n o m e r s u p p l y are two l i m i t i n g factors i n cell w a l l l i g n i f i c a t i o n . B u t peroxidase a v a i l a b i l i t y m i g h t not be i n itself a l i m i t i n g factor since the e n z y m e is present i n most cell w a l l s . F o r i n s t a n c e , i f the h y p o t h e s i s of peroxidase p o l y f u n c t i o n a l i t y was p r o v e d to be t r u e , l i m i t i n g factors w o u l d be the molecules c o n t r o l l i n g the c o n f o r m a t i o n a l changes necessary t o i n d u c e specific r e c o g n i t i o n of c i n n a m y l alcohols. A l t e r n a t i v e l y , c y t o p l a s m i c c o m p a r t m e n t s , e s p e c i a l l y the vacuoles, c o n t a i n peroxidases w i t h a w i d e range of affinities. S o m e of t h e m are even able to o x i d i z e s y r i n g a l d a z i n e (7,9). T h e y c o u l d c o n s t i t u t e a storage p o o l of e n z y m e , a l l o w i n g r a p i d responses to m e t a b o l i c or e n v i r o n m e n t a l changes.
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a n d substrate c o n c e n t r a t i o n (S). T h e substrates were i s o p r o p y l a m i n e salts f r o m /?-fluoroferulic a c i d ( A a n d B ) a n d ferulic a c i d ( C a n d D ) . A a n d C : x y l e m isoperoxidase Χχ, Β a n d D : x y l e m isoperoxidase X 2 .
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PLANT C E L L W A L L POLYMERS
Literature Cited
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