Biogenesis and Localization of Polymethylated Flavonoids in Cell

instead a variety of tri- to penta-O-methylated flavonol glucosides. These highly lipophilic metabolites were found associated with the plant cell wal...
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Chapter 8

Biogenesis and Localization of Polymethylated Flavonoids in Cell Walls of Chrysosplenium americanum Ragai Ibrahim, Lilian Latchinian, and Louise Brisson Plant Biochemistry Laboratory, Department of Biology, Concordia University, Montreal, Quebec H3G 1M8, Canada

The semiaquatic species, Chrysosplenium americanum (Saxifragaceae), which lacks lignin, accumulates a number of tri- to penta-O-methylated flavonol glucosides instead. Their enzymatic synthesis is catalyzed by five position-specific methyltransferases, which exhibited distinct p H optima, pI values, cation requirements and preference for flavonol aglycones or glucosides as substrates. O-Glucosylation of methylated flavonols at 2'and 5'-oppositions is mediated by two distinct glucosyltransferases, which were resolved by affinity chromatography on UDP-glucuronic acid agarose and Brown 10X dye ligand. Kinetic analysis of both groups of enzymes indicates that this multistep pathway is subject to tight control, and that the kinetic constants regulate the rate of synthesis of the final products. T h e use of electron microscopy, immunofluorescence and immunocytochemical techniques unequivocally indicate that these highly lipophilic metabolites are associated with the walls of epidermal and mesophyll cells. It was more t h a n 20 years ago t h a t h y d r o x y c i n n a m i c acids were r e p o r t e d (1) t o be esterified i n appreciable a m o u n t w i t h the u n l i g n i f i e d cell walls o f grasses a n d other p l a n t species. M o n o c o t a r a b i n o x y l a n s ( a hemicellulose f r a c t i o n ) have been reported t o be s u b s t i t u t e d w i t h p - c o u m a r a t e , ferulate a n d p - h y d r o x y b e n z o a t e (2,3), a n d y i e l d e d o n e n z y m a t i c h y d r o l y s i s feruloyl a r a b i n o s y l x y l o s e . A l k a l i n e h y d r o l y s i s o f grass cell walls gave d i f e r u l i c a c i d ( 2 , 4 - 6 ) , t h e o x i d a t i v e c o u p l i n g p r o d u c t o f feruloyl residues. P e c t i n s f r o m s p i n a c h c u l t u r e cell walls c o n t a i n ferulate a n d p - c o u m a r a t e ( 7 , 8 ) , w h i c h o n p a r t i a l h y d r o l y s i s give t w o feruloyl dissacharides, 4-0-(6-feruloyl-/?D-galactosyl)-D-galactose and 3-0-(3-feruloyl-a-L-arabinosyl)-L-arabinose 0097-6156/89/0399-0122$06.00/0 C 1989 American Chemical Society

8.

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(9) a n d a s m a l l a m o u n t of d i f e r u l a t e . T h e c o m m o n occurrence o f p h e n o l i c a c i d sugar esters i n a s s o c i a t i o n w i t h cell walls is not s u r p r i s i n g , since these are k n o w n n a t u r a l p l a n t c o n s t i t u e n t s (10). O n the other h a n d , the cell w a l l g l y c o p r o t e i n , e x t e n s i n contains the o x i d a t i v e l y c o u p l e d d i m e r , i s o d i t y rosine (11) a n d a t r i m e r of t y r o s i n e (12) w h i c h have been p r o p o s e d (13) to crosslink w i t h two or three p o l y p e p t i d e c h a i n s of e x t e n s i n . I n contrast w i t h the above m e n t i o n e d p h e n o l i c residues, w h i c h m a y be Η-bonded or covalently l i n k e d w i t h different cell w a l l f r a c t i o n s , a n o t h e r group of phenolic compounds-the simple flavonoids-may be secreted o n p l a n t surfaces as g u m m y or farinose exudates. These were first r e p o r t e d i n p o p l a r (14) a n d are now k n o w n to o c c u r i n m a n y w o o d y a n d herbaceous species, i n c l u d i n g ferns (15). O n the other h a n d , the s e m i - a q u a t i c species, Chrysosplenium americanum (Saxifragaceae) w h i c h l a c k s l i g n i n , synthesizes i n s t e a d a v a r i e t y of t r i - to p e n t a - O - m e t h y l a t e d flavonol glucosides. T h e s e h i g h l y l i p o p h i l i c m e t a b o l i t e s were f o u n d associated w i t h the p l a n t cell w a l l s , a l t h o u g h the n a t u r e o f t h i s a s s o c i a t i o n is yet to be d e t e r m i n e d . F l a v o n o i d c o m p o u n d s are n a t u r a l p l a n t c o n s t i t u e n t s of w i d e d i s t r i ­ b u t i o n i n n a t u r e ; a m o n g w h i c h are the flower a n d f r u i t p i g m e n t s ( a n t h o c y a n i n s ) a n d the yellow p i g m e n t s (flavones a n d flavonols), f o u n d i n a l l p l a n t p a r t s . These c o m p o u n d s share a c o m m o n b i o s y n t h e t i c o r i g i n ; b e i n g f o r m e d of two p h e n o l i c r i n g systems A a n d B , w h i c h are d e r i v e d f r o m acetate a n d c i n n a m a t e , respectively, a l t h o u g h they differ i n the o x i d a t i o n level of the h e t e r o c y c l i c r i n g C (see s t r u c t u r e b e l o w ) . F l a v o n e s a n d flavonols, w h i c h o c c u r n a t u r a l l y as glycosides, m a y be O - m e t h y l a t e d or O - g l u c o s y l a t e d at one or several p o s i t i o n s o n the flavonoid r i n g s y s t e m . P a r t i a l l y a n d f u l l y m e t h y l a t e d flavonoids are n o w considered to be w i d e l y d i s t r i b u t e d i n the p l a n t k i n g d o m ( 1 6 , 1 7 ) . B o t h e n z y m a t i c reactions are believed to p l a y an i m p o r t a n t role i n the d e t o x i f i c a t i o n of reactive h y d r o x y l groups a n d hence, their c o m p a r t m e n t a t i o n w i t h i n p l a n t cells a n d tissues (18).

E n z y m a t i c O - m e t h y l a t i o n of flavonoids, w h i c h is c a t a l y z e d b y Omethyltransferases ( E . C . 2.1.1.6-) involves the transfer of the m e t h y l g r o u p of a n a c t i v a t e d m e t h y l d o n o r , 5 - a d e n o s y l - L - m e t h i o n i n e , to the h y d r o x y l g r o u p of a flavonoid acceptor w i t h the f o r m a t i o n o f the c o r r e s p o n d i n g methylether and 5-adenosyl-L-homocysteine. T h e latter product is, in

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most cases, a c o m p e t i t i v e i n h i b i t o r of the e n z y m e r e a c t i o n (19). A n u m b e r of flavonoid-specific O-methyltransferases has recently been c h a r a c t e r i z e d ; a m o n g w h i c h are those w h i c h a t t a c k p o s i t i o n s 3' of flavones a n d flavonols (20-22), 3 7 5 ' of a n t h o c y a n i n s (23), 4' of isoflavones (24) a n d flavones (25), 5 of isoflavones (26), 7 of flavonols (27,28) a n d C-glycoflavones (29) a n d 8 of flavonols ( 3 0 , 3 1 ) . It s h o u l d be n o t e d , however, t h a t these enzymes c a t a l y z e d single m e t h y l a t i o n steps, a n d d i d not accept p a r t i a l l y m e t h y l a t e d substrates for further O - m e t h y l a t i o n . T h e c o m m o n occurrence of p a r t i a l l y m e t h y l a t e d flavonoids ( 1 6 , 1 7 ) , such as those of C. amencanum ( F i g u r e 1), raised the question as to whether m u l t i p l e m e t h y l transfers were c a t a l y z e d b y one or several position-specific O-methyltransferases! E n z y m a t i c g l u c o s y l a t i o n , o n the other h a n d , is m e d i a t e d b y O glucosyltransferases ( E . C . 2.4.1-) a n d involves the transfer of the g l u c o s y l m o i e t y of a nucleotide d i p h o s p h a t e sugar to the h y d r o x y l groups of a phen o l / f l a v o n o i d acceptor w i t h the f o r m a t i o n of the c o r r e s p o n d i n g glucoside a n d the nucleotide d i p h o s p h a t e . T h e l a t t e r m a y also act as c o m p e t i t i v e i n h i b i t o r of the e n z y m e reaction (32). A l t h o u g h glucosyltransferases are k n o w n to be substrate-specific a n d p o s i t i o n - o r i e n t e d (33), i t is not k n o w n whether g l u c o s y l a t i o n of the less c o m m o n 2' a n d 5' p o s i t i o n s of flavonoids (e.g., c o m p o u n d s I a n d I I I , respectively, F i g u r e 1) is c a t a l y z e d b y one or two d i s t i n c t enzymes. T h e present work describes the m u l t i e n z y m e s y s t e m w h i c h is i n v o l v e d i n the m e t h y l a t i o n - g l u c o s y l a t i o n sequence of Chrysosplenium flavonoids. T h e l a t t e r tissue is a n i d e a l e x p e r i m e n t a l s y s t e m since i t a c c u m u l a t e s s i x , t r i - to p e n t a - O - m e t h y l a t e d flavonol glucosides. T w o of these, I a n d I I , are derivatives of 2 ' - s u b s t i t u t e d q u e r c e t i n ( 3 , 5 , 7 , 4 ' , 5 ' - p e n t a - h y d r o x y f l a v o n e ) ; two others, III a n d I V , are 6 - s u b s t i t u t e d q u e r c e t i n (quercetagetin) d e r i v a tives; whereas the r e m a i n i n g two c o m p o u n d s , V a n d V I , are 2 ' - s u b s t i t u t e d quercetagetin ( F i g u r e 1). W h e r e a s the first p a i r of flavonoids is g l u c o s y l a t e d at the less c o m m o n 2 ' - p o s i t i o n , a l l the others are 5 ' - 0 - g l u c o s i d e s . N o n e of the lower m e t h y l a t e d intermediates of the p a t h w a y a c c u m u l a t e i n t h i s tissue, a l t h o u g h they can be synthesized e n z y m a t i c a l l y in vitro (34). D u e to the h i g h l y l i p o p h i l i c , a n d p a r t i a l l y h y d r o p h i l i c , n a t u r e of these m e t a b o l i t e s it was considered of interest to s t u d y their i n t r a c e l l u l a r l o c a l i z a t i o n i n t h i s tissue. Biogenesis of Chrysosplenium Flavonoids. T r a c e r e x p e r i m e n t s u s i n g [2C ] c i n n a m a t e a d m i n i s t e r e d to y o u n g shoots resulted i n l a b e l i n g of the s i x m e t h y l a t e d flavonol glucosides w i t h i n 5 to 10 m i n pulse ( F i g u r e 2), b u t none of the low m e t h y l a t e d intermediates. F u r t h e r m o r e , the p a r t i a l l y p u rified e n z y m e p r e p a r a t i o n s c a t a l y z e d the m e t h y l a t i o n of q u e r c e t i n , b u t not q u e r c e t a g e t i n , to its 3-mono-, 3 , 7 - d i - a n d 3 , 7 , 4 - t r i m e t h y l derivatives (3436), as w e l l as the g l u c o s y l a t i o n of p a r t i a l l y m e t h y l a t e d i n t e r m e d i a t e s to their c o r r e s p o n d i n g glucosides (37). These results suggested the existence, i n t h i s tissue, of the enzyme c o m p l e m e n t involved i n the biosynthesis of these m e t a b o l i t e s a n d p r o m p t e d the c h a r a c t e r i z a t i o n of the i n d i v i d u a l e n zymes of the pathway. 1 4

/

F i g u r e 1. P o s t u l a t e d p a t h w a y for the e n z y m a t i c synthesis of p o l y m e t h y l a t e d flavonol glucosides i n Chrysosplenium americanum: G T , O-glucosyltransferase; O H , h y d r o x y l a s e ; O M T , O - m e t h y l transferase.

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T h e Multistep Methylation

POLYMERS

Sequence

Purification of 0-Methyltransferases. Using conventional, open-column chromatography alternatively with a fast protein liquid chromatography ( F P L C ) system, we were able to purify five distinct, position-specific methyltransferases (38,39). T h e last step of purification was achieved by chromatofocusing on a Mono Ρ analytical column (40) using a gradient between p H 6 and p H 4 (Figure 3). T h e extent of purification of these enzymes varied between 85 to 164-fold using conventional column and 400 to 650-fold using the F P L C system (Table I). Table I. Properties of Chrysosplenium Property Purification (-fold) Open column HPLC p H optimum p i value M o l . W t . (Kd) K m (SAM), / i M K m (Flav), μΜ K i (SAH), μΜ K i (Me-Flav), μΜ

Methyltransferases

3-

6-

7-

4'-

2'/5'

85 650 4.8 4.0 57 114 12 4.5 128

92 400 9.0 5.2 57 51 18 16 167

6 460 8.2 4.8 57 65 7 10 15

164 460 8.8 5.0 57 130 15 4.4 10

123 420 7.0 4.6 57 100 2

Substrate Specificity of O-Methyltransferases. A flavonol with 4', 5'hydroxylation pattern, such as quercetin but not quercetagetin, is be­ lieved to be the first methyl acceptor in this pathway. T h e highly pu­ rified enzymes (Figure 3) exhibited strict position specificity for po­ sitions 3 of quercetin (3-methyltransferase), 7 of 3-methylquercetin (7methyltransferase), 4' of 3,7-dimethylquercetin (4'-methyl transferase), 6 of 3,7,4'-trimethylquercetagetin (6-methyltransferase), 5' of 5,2',5'-trihydroxy-3,7,4 -trimethoxyflavone-2 -glucoside and 2'-position of 5,2', 5'trihydroxy-3,6,7,4'- tetramethoxyflavone-5'- glucoside ^'-/ô'-methyltransferase). It is interesting to note that, in contrast with the earlier enzymes of the pathway which utilized aglycones as substrates, the two later methylation steps (2'- and 5'-positions) took place at the glucoside level (Fig. 1), and were catalyzed equally well by the same protein preparation (39,40). However, in view of the strict position specificity of the former enzymes (3-, 6-, 7- and 4 -methyltransferases), it can be assumed that the two later methylation steps of the pathway may be mediated by two distinct enzymes which remain to be resolved. None of these enzymes accepted phenylpropanoids, flavones, dihydroflavonols, or any of their glucosides. Both substrate and position specificities of these enzymes indicate a coordinated sequence of methyl transfers to quercetin —• 3-methylquercetin —*· 3,7dimethyl-quercetin —• 3,7,4'-trimethylquercetin. After hydroxylation of ,

/

,

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Origin

F i g u r e 2. P h o t o g r a p h of a n a u t o r a d i o g r a m of the c h r o m a t o g r a p h e d leaves a d m i n i s t e r e d [ C ] c i n n a m a t e for 10 m i n . C o m p o u n d s I to V I ( F i g u r e 1) are the final flavonoid metabolites w h i c h a c c u m u l a t e i n t h i s tissue. 14

10

20

Fraction

30

40

50

number

F i g u r e 3. E l u t i o n profile of five O-methyltransferases after c h r o m a t o f o c u s ­ i n g o n a n a n a l y t i c a l M o n o - P c o l u m n , u s i n g a g r a d i e n t between p H 6 a n d p H 4.

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the l a t t e r at p o s i t i o n 2' a n d its subsequent g l u c o s y l a t i o n , i t is f u r t h e r m e t h y ­ l a t e d t o its 5 ' - m e t h y l derivative ( c o m p o u n d I I ) . 3 , 7 , 4 - t r i m e t h y l q u e r c e t i n m a y also be h y d r o x y l a t e d at p o s i t i o n 6, f u r t h e r m e t h y l a t e d at t h a t p o s i t i o n ( c o m p o u n d I I I ) , t h e n g l u c o s y l a t e d at 5' ( c o m p o u n d I V ) . T h e l a t t e r , after f u r t h e r h y d r o x y l a t i o n at the 2 ' - p o s i t i o n ( c o m p o u n d V ) is f i n a l l y m e t h y l a t e d t o c o m p o u n d V I ( F i g u r e 1). ,

Properties of 0-Methyltransferases. T h e different enzymes e x h i b i t e d d i s ­ t i n c t p i values a n d p H o p t i m a ( T a b l e I), a l t h o u g h t h e y h a d s i m i l a r m o l e c u ­ l a r weights. U n l i k e the other enzymes of the m e t h y l a t i o n sequence, the 6-methyltransferase e x h i b i t e d absolute requirement for M g ions, whose a c t i v a t i o n was s a t u r a b l e a n d was i n h i b i t e d b y E D T A . T h e different Omethyltransferases were i n h i b i t e d b y 1 m M of the S H g r o u p reagents, pchloromercuri-benzoate and N-ethylmaleimide to various extent. T h e a d d i ­ t i o n of 14 m M 2 - m e r c a p t o e t h a n o l p a r t i a l l y prevented t h i s i n h i b i t i o n (38). Kinetics of 0-Methylation. T h e steady state k i n e t i c a n a l y s i s of these e n ­ zymes ( 4 1 , 4 2 ) was consistent w i t h a sequential ordered r e a c t i o n m e c h a ­ nism, i n which 5-adenosyl-L-methionine and 5-adenosyl-L-homocysteine were l e a d i n g r e a c t i o n p a r t n e r s a n d i n c l u d e d a n a b o r t i v e E Q B c o m p l e x . F u r t h e r m o r e , a l l the methyltransferases s t u d i e d e x h i b i t e d c o m p e t i t i v e p a t ­ terns between S - a d e n o s y l - L - m e t h i o n i n e a n d i t s p r o d u c t , whereas the other p a t t e r n s were either n o n c o m p e t i t i v e or u n c o m p e t i t i v e . W h e r e a s the 6m e t h y l a t i n g e n z y m e was severely i n h i b i t e d by its respective flavonoid s u b ­ strate at concentrations close t o K m , the other enzymes were less affected. T h e low i n h i b i t i o n constants of 5 - a d e n o s y l - L - h o m o c y s t e i n e ( T a b l e I) s u g ­ gests t h a t earlier enzymes of the p a t h w a y m a y regulate the rate of synthesis of the final p r o d u c t s . Glucosylation of Partially Methylated

Flavonols

T h e fact t h a t Chrysosplenium flavonoids are g l u c o s y l a t e d at the less c o m ­ m o n 2 ' - a n d o p p o s i t i o n s p r o m p t e d a n i n v e s t i g a t i o n as t o whether b o t h g l u c o s y l a t i o n steps are m e d i a t e d b y one or t w o d i s t i n c t enzymes. Purification of 2'-/5'-0-Glucosyltransferase. P r e v i o u s studies i n t h i s l a b o ­ r a t o r y (37) have d e m o n s t r a t e d the existence, i n t h i s tissue, o f a novel r i n g B-specific O-glucosyltransferase. T h i s e n z y m e a t t a c k e d either 2'- or 5'p o s i t i o n o f p a r t i a l l y m e t h y l a t e d flavonols a n d required t w o , para-oriented s u b s t i t u e n t s o n r i n g Β for o p t i m u m a c t i v i t y (e.g., aglycones of I I a n d V I , F i g u r e 1). T h e fact t h a t the 2'- a n d 5 ' - g l u c o s y l a t i n g a c t i v i t i e s c o u l d not be separated b y either c o n v e n t i o n a l c h r o m a t o g r a p h y o n several c o l u m n s (37) or b y F P L C (43), seemed t o i n d i c a t e t h a t b o t h g l u c o s y l a t i o n s m a y be c a t a l y z e d b y one e n z y m e . However, u n l i k e other glucosyltransferases (32) the Chrysosplenium e n z y m e b i n d s U D P (44), the second p r o d u c t of the r e a c t i o n . T h i s k i n e t i c p r o p e r t y allowed b i n d i n g the e n z y m e t o a U D P g l u c u r o n i c a c i d agarose affinity s u p p o r t (43), a l t h o u g h i t d i d not b i n d to U D P - a g a r o s e . T h e glucosyltransferase a c t i v i t y was e l u t e d at a p p r o x i m a t e l y 60 m M K C 1 , t h e n desalted before b e i n g a p p l i e d to B r o w n 1 0 X dye l i g a n d at p H 6.5. E l u t i o n o f the e n z y m e p r o t e i n f r o m the l a t t e r c o l u m n was p e r ­ f o r m e d u s i n g a l i n e a r p H - s a l t g r a d i e n t , a n d resulted i n the s e p a r a t i o n of

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the 2'- and 5'-activities at p H values of 7.8 and 7.3, respectively (Figure 4). The combined purification steps resulted in an increase in specific activity of 1200-fold (43). Each of the purified fractions gave a single flavonoid glucoside when assayed against the respective aglycone, as shown by autoradiography of the reaction products (Figure 4, insert). These results indicate that glucosylation of the 2'-and 5'-positions of flavonoids is catalyzed by two distinct enzymes. It should be pointed out that, in contrast with the enzymes of primary metabolism (e.g., photosynthesis, respiration, etc.), those catalyzing the synthesis of secondary metabolites usually occur in very low abundance, and are therefore, very difficult to purify to homogeneity especially for the purpose of raising antibodies. However, the recent advances in immunization and selection techniques made it possible to overcome the need for homogeneous protein in order to raise monoclonal antibodies. Immunological Evidence for 2*-O-Glucosyltransferase. Murine monoclonal antibody to the partially purified enzyme was produced by an in vitro immunization technique (45) of B a l b / c mice spleen cells, followed by fusion with mouse myeloma cells. Screening culture supernatants of the resulting hybridomas by an enzyme-linked immunosorbent assay ( E L I S A ) revealed the presence of two highly immunoreactive IgM-secreting clones, C3-2 and C7-1 (Table II) (46). Only the former clone displayed > 50% inhibition of the 2'-glucosyltransferase activity, whereas neither antibodies C3-2 nor C 7 1 inhibited the 5'-activity (Figure 5). These results clearly demonstrate the existence of an immunologically distinct flavonol 2'-0-glucosyl transferase. Furthermore, the native form of this enzyme was essential for recognition by the C3-2 antibody, hence, immunoreactive bands on Western blots (Figure 5, insert) could only be visualized following n a t i v e - P A G E , and not S D S - P A G E . Further work is aimed at selecting a 5'-specific antibody in order to demonstrate, unequivocally, the involvement of two distinct 2'- and 5 -glucosyltransferases. /

Table II. Specific Fusion Efficiency Ig-Producing Wells (% of total) Fusion

IgM

IgG

Specific Immunoreactive Wells (% of IgM-Secretors)

Day 5 Day 7

50.5 45.9

3.6 0.5

22.2 18.6

Properties of the Glucosyltransferase. Except for its strict substrate specificity, the properties of this enzyme were similar to those of other flavonoid glucosyltransferases (32,33) and are listed in Table III. Kinetics of Glucosylation. T h e detailed kinetic analysis of the partially purified enzyme (44) was consistent with an ordered bi bi mechanism, where UDP-glucose binds to the enzyme first, followed by the flavonoid aglycone

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V o l u m e (ml) F i g u r e 4. E l u t i o n profile of the 2'- a n d 5 ' - 0 - g l u c o s y l t r a n s f e r a s e s after c h r o m a t o g r a p h y o n B r o w n 1 0 X agarose c o l u m n u s i n g p H - s a l t gradient. Insert: a u t o r a d i o g r a p h e d e n z y m e reaction p r o d u c t s .

I

1

I

I

I

0.25

0.5

0.75

1.0

I

MCAB DILUTION

F i g u r e 5. I m m u n o r e m o v a l ( i n h i b i t i o n ) of 2'-glucosyltransferase a c t i v i t y b y different concentrations of m o n o c l o n a l a n t i b o d y C 3 - 2 . Insert: W e s t e r n b l o t of the n a t i v e e n z y m e after P A G E (left, c o n t r o l ; r i g h t , b l o t w i t h C 3 - 2 a n t i body).

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Table III. Properties of Chrysosplenium Property Purification (-fold) p H optimum p i value M o l . W t . (Kd) K m (UDPglc) μΜ K m (Flav.) μΜ K i ( U D P ) μΜ K i (Flav. gluc.) μΜ

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2'-

5'

1200 8.0 5.2 42 250 5 25 1000

1200 8.0 5.2 42 250 10 20 1000

and the release of the glucoside followed by U D P . T h e latter was a com­ petitive inhibitor with respect to UDP-glucose and noncompetitive with respect to both UDP-glucose and the flavonoid substrate. T h e high i n ­ hibition constant of the glucosylated product, as compared with those of the substrate and co-substrate (Table III) indicates that glucosylation of the partially methylated flavonoids is not inhibited by the products formed (44), and is consistent with the accumulation of compounds I, II, V and V I (Figure 1) as the major flavonoid constituents of this tissue. Regulation of Flavonoid Synthesis in C. americanum. Biosynthesis of methylated flavonol glucosides seems to be under tight regulation, not only by the substrate specificity of the enzymes involved, but also by other fac­ tors, among which are: (a) the strict position specificity of these enzymes towards their hydroxylated or partially methylated substrates; (b) the ap­ parent difference in microenvironment of the different methyl-transferases, whereby those earlier in the pathway utilized aglycones whereas later en­ zymes accepted only glucosides as substrates; (c) the subtle characteris­ tic differences in methyl-transferases with respect to their p H optima, p i values and requirement for M g ions, despite their similar molecular size; (d) the sequential ordered mechanism of all the enzymes involved in the methylation-glucosylation sequence of this pathway. Another important aspect of regulation derives from the kinetic analysis of the enzymes stud­ ied. This is demonstrated by the similarity of their kinetic mechanisms and their regulation by a specific range of substrate and product concentrations (41,42). Despite the fact that the methylating enzymes had K m values in the same range as those of glucosylation, however, the K i values for the latter reaction were in the m M range as compared with the μΜ values for methylation (Tables I and III), and are consistent with glucoside accumula­ tion as the final products. Another means of regulation of the methylation sequence involves the differential affinities of the different enzymes for 5adenosyl-L-methionine and 5-adenosyl-L-homocysteine (41,42). Whereas the four methyltransferases studied exhibited similar affinities for their re­ spective flavonoid substrates (Table I), the affinity for the methyl donor 5adenosyl-L-methionine was similar for the 3- and 4'-0-methyltransferases, while those attacking positions 6 and 7 were two times greater. Further-

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m o r e , the former enzymes were subject to i n h i b i t i o n b y low c o n c e n t r a t i o n s of 5 - a d e n o s y l - L - h o m o c y s t e i n e , since the a p p a r e n t K i for the l a t t e r t w o e n zymes was 25 t i m e s smaller t h a n the K m for S - a d e n o s y l - L - m e t h i o n i n e , as c o m p a r e d w i t h 3 a n d 6 times for the 6- a n d 7 - m e t h y l transferases, respect i v e l y ( T a b l e I). These characteristics suggest t h a t the enzymes earlier i n the m e t h y l a t i o n sequence m a y regulate the rate of synthesis o f the final products. Localization Studies. L o c a l i z a t i o n of flavonoid c o m p o u n d s has u s u a l l y been s t u d i e d u s i n g c y t o c h e m i c a l m e t h o d s , i n c l u d i n g the f o r m a t i o n of p r e c i p i tates w i t h s u i t a b l e reagents for nonfluorescent c o m p o u n d s ( 1 8 , 3 3 a n d refs. t h e r e i n ) , as w e l l as u l t r a v i o l e t fluorescence m i c r o s c o p y for fluorescent c o m p o u n d s . D e s p i t e the variety o f techniques used i n the s e p a r a t i o n of p l a n t tissues for l o c a l i z a t i o n of m e t a b o l i t e s , it is difficult to interpret the d a t a due to the v a r y i n g degree of p u r i t y of the isolated t i s s u e / o r g a n e l l e , or its possible c o n t a m i n a t i o n w i t h the m e t a b o l i t e s released d u r i n g i s o l a t i o n . S u c h p r o b l e m s have been c o m p o u n d e d b y the lack of specific h i s t o c h e m i c a l tests for the detection o f p h e n o l i c / f l a v o n o i d c o m p o u n d s , a n d t h e i r s o l u b i l i t y i n the o r g a n i c solvents n o r m a l l y used for microscopic p r e p a r a t i o n s . T h e r e fore, we have used three different strategies for the l o c a l i z a t i o n , in situ, of Chrysosplenium p o l y m e t h y l a t e d flavonol glucosides. Electron Microscopy. U s i n g caffeine as prefix a n d v i s u a l i z i n g agent (47 a n d refs. cited therein) allowed us to s t u d y the u l t r a s t r u c t u r a l features of flavonoid a c c u m u l a t i o n i n t h i s tissue. These studies (48) revealed the presence of electron-dense deposits w i t h i n the walls of leaf e p i d e r m a l a n d m e s o p h y l l cells ( F i g u r e 6 A ) . V a r i o u s m e m b r a n e profiles a n d associated vesicles i n the p e r i p l a s m i c area were also filled w i t h d a r k l y s t a i n e d m a t e r i a l . T h e fact t h a t most of these vesicles were fused w i t h the p l a s m a l e m m a ( F i g ure 6 A ) suggested the secretory n a t u r e o f these cells (47). T h e r e was no evidence to i n d i c a t e t h a t the G o l g i a p p a r a t u s was i n v o l v e d i n p a c k a g i n g or c h a n n e l i n g of flavonoids. F u r t h e r m o r e , the cell w a l l flavonoid deposits ( F i g ure 6 A ) c o u l d be leached out by d i p p i n g leaf segments i n organic solvents for 1-2 second intervals (48). H P L C a n a l y s i s of these effusates i n d i c a t e d the recovery of the m a j o r flavonoid constituents. These observations are consistent w i t h the l i p o p h i l i c n a t u r e of the h i g h l y m e t h y l a t e d flavonol g l u cosides a n d t h e i r l o c a l i z a t i o n w i t h i n the walls of e p i d e r m a l a n d m e s o p h y l l cells of t h i s tissue. Immunofluorescence. T h i s is a sensitive, h i g h l y specific technique w h i c h has recently been used for the l o c a l i z a t i o n of large molecules, such as storage proteins or enzymes. T o our knowledge, there has been no i n f o r m a t i o n on the use of this technique for flavonoid l o c a l i z a t i o n . T h i s m a y be due to the difficulty i n r a i s i n g antibodies against r e l a t i v e l y s m a l l molecules (ca. < 5000 d a l t o n ) . C o m p o u n d I ( F i g u r e 1) was conjugated to b o v i n e s e r u m a l b u m i n b y the d i a z o reaction (49) a n d was used to raise a n t i b o d y i n r a b b i t s (50). T h i s a n t i b o d y was f o u n d to be specific for the 2'-glucosides of t r i - a n d tetramethoxyflavones I a n d II. However, there was some cross rea c t i v i t y against the pentamethoxyflavone-5'-glucoside ( c o m p o u n d V I ) , b u t none w i t h q u e r c e t i n or any of its t r i - or t e t r a m e t h y l derivatives (50). T h i s

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Figure 6. A: Photomicrograph ( χ 51,000) of caffeine treated leaf epidermal cell showing electron-dense deposits on cell wall and membrane vesicles fusing with the plasmalemma (arrows). B: Immunofluorescence labeling of flavonoids in cell walls of leaf epidermal strips (arrows) and autofluorescent stomata (x 62.5). C: Immunogold labeling of the walls of a mesophyll cell (left, χ 41,000). Ch, chloroplast; EC, epidermal cell; G, Golgi; IS, intercellular space; MC, mesophyll cell; (right, control χ 19,500).

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a n t i b o d y was a p p l i e d w i t h a n i n d i r e c t immunofluorescence technique (51) u t i l i z i n g fluorescein i s o t h i o c y a n a t e - l a b e l e d goat a n t i r a b b i t a n t i b o d y w i t h leaf e p i d e r m i s , cross sections a n d p r o t o p l a s t s . T h e results ( F i g u r e 6 B ) i n d i c a t e d t h a t flavonoid a c c u m u l a t i o n o c c u r r e d m a i n l y i n the walls o f e p i d e r m a l cells a n d , to a m u c h lesser extent, i n m e s o p h y l l cell w a l l s . T h e weak fluorescence observed i n the vacuoles of p r o t o p l a s t s suggested a m i n o r role of t h i s c o m p a r t m e n t i n the a c c u m u l a t i o n process (51). Immunocytochemistry. F u r t h e r u n e q u i v o c a l evidence for the site of flavonoid a c c u m u l a t i o n was o b t a i n e d u s i n g the p r o t e i n A - g o l d p o s t - e m b e d d i n g technique (52), coupled w i t h t r a n s m i s s i o n electron m i c r o s c o p y (53). A n t i b o d y - s p e c i f i c l a b e l i n g was observed m a i n l y o n the walls o f epiderm a l a n d m e s o p h y l l cells ( F i g u r e 6 C ) . F u r t h e r m o r e , there was s i g n i f i c a n t a m o u n t o f l a b e l i n g associated w i t h the p l a s m a l e m m a , b u t none w i t h other organelles such as the e n d o p l a s m i c r e t i c u l u m , G o l g i or chloroplasts. T h e s e results p r o v i d e s t r o n g evidence for the l o c a l i z a t i o n o f p a r t i a l l y m e t h y l a t e d flavonol glucosides i n the cell walls of t h i s tissue. Significance of Flavonoid A c c u m u l a t i o n i n Cell Walls W h e r e a s the a c c u m u l a t i o n of flavonoids i n p l a n t cell walls m a y be difficult to e x p l a i n , however, i t m a y be considered as a means of e l i m i n a t i n g such c y t o t o x i c agents f r o m the cell s y m p l a s t . S u c h site for flavonoid a c c u m u l a t i o n m a y also be considered as a means of p r o t e c t i o n against pathogens, predators a n d u l t r a v i o l e t r a d i a t i o n (54), especially i n the absence of l i g n i fied tissues, as i n the case of Chrysosplenium. Conclusions T o o u r knowledge, t h i s is the first r e p o r t e d instance where flavonoid c o m p o u n d s have been f o u n d i n a s s o c i a t i o n w i t h p l a n t cell w a l l s . W h e r e a s the enzymes i n v o l v e d i n the biosynthesis of these w a l l constituents have a l w a y s been recovered i n the cytosolic f r a c t i o n , it is not k n o w n whether they are a c t u a l l y soluble, or easily s o l u b i l i z e d enzymes. It is t e m p t i n g to p o s t u l a t e t h a t flavonoid synthesis i n Chrysosplenium takes place o n the surface o f a n aggregated, m e m b r a n e associated (e.g. the E R ) m u l t i e n z y m e s y s t e m (55), where the component enzymes m a y be loosely associated or held together b y non-covalent b o n d s . Despite the unsuccessful a t t e m p t s to isolate s u c h an aggregate, however, several lines of evidence ( b i o s y n t h e t i c , e n z y m a t i c a n d k i n e t i c ) , m e n t i o n e d above, tend to s u p p o r t t h i s concept. D u e to t h e i r c y t o t o x i c i t y a n d h i g h l y l i p o p h i l i c n a t u r e , these m e t a b o l i t e s m a y be sequestered i n the f o r m of 'flavonoid vesicles,' w h i c h have been observed (48) to fuse w i t h the p l a s m a l e m m a for the discharge o f t h e i r contents w i t h i n the cell walls. T h e fact t h a t these m e t a b o l i t e s were r e a d i l y recovered f r o m leaf effusates suggests t h a t they m a y be adsorbed o n cell w a l l p r o t e i n s . S u b c e l l u l a r l o c a l i z a t i o n of the enzymes c a t a l y z i n g t h i s p a t h w a y s h o u l d p r o v i d e s u p p o r t i n g evidence for this proposed m o d e l . W e are w o r k i n g a l o n g these lines. Acknowledgments W o r k c i t e d f r o m the senior a u t h o r ' s l a b o r a t o r y has been s u p p o r t e d b y o p e r a t i n g a n d e q u i p m e n t grants f r o m the N a t u r a l Sciences a n d E n g i n e e r i n g

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RECEIVED March 17, 1989